ABSTRACT
Predisposition to rheumatoid arthritis (RA) is thought to be associated with HLA-DR1, -DR4, and -DR10. However, many epidemiological observations are better explained by a model in which the DQ alleles that are linked to these DR alleles, i.e., DQ5, DQ7, and DQ8, predispose to RA, while certain DR alleles have a dominant protective effect. All protective DRB1 alleles, e.g., *0402, *1301, and *1302, encode a unique motif, (70)DERAA(74). The protection may be explained by the presentation of DRB1-derived peptides by DQ to immunoregulatory T cells, because it was demonstrated in various autoimmune disease models that T cell responses to certain self-Ags can be involved in disease suppression. The aim of this study was to analyze whether peptides carrying the DERAA motif are naturally processed by human APC and presented in the context of the RA-predisposing DQ. Using a synthetic peptide carrying the DRB1*0402-derived sequence (65)KDILEDERAAVDTYC(79), we generated DERAA peptide-specific DQ-restricted T cell clones (TCC) from a DQ8 homozygous individual carrying DERAA-negative DR4 alleles. By analyzing the proliferation of these TCC, we demonstrated natural processing and presentation of the DERAA sequence by the APC of all the individuals (n = 12) carrying a DERAA-positive DRB1 allele and either DQ8 or the DQ8-related DQ7. Using a panel of truncated synthetic peptides, we identified the sequence (67)(I)LEDERAAVD(TY)(78) as the minimal determinant for binding to DQ8 and for recognition by the TCC. These findings support a model in which self-MHC-derived peptide can modulate predisposition to autoimmune disease in humans.
Subject(s)
Antigen Presentation , Arthritis, Rheumatoid/immunology , HLA-DR Antigens/immunology , HLA-DR Antigens/metabolism , Peptide Fragments/immunology , Peptide Fragments/metabolism , Alleles , Amino Acid Sequence , Antigen Presentation/genetics , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/metabolism , Cell Line , Clone Cells , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/immunology , Genetic Predisposition to Disease , HLA-DQ Antigens/genetics , HLA-DQ Antigens/immunology , HLA-DQ Antigens/metabolism , HLA-DR Antigens/genetics , HLA-DRB1 Chains , Humans , Lymphocyte Activation/genetics , Molecular Sequence Data , Peptide Fragments/genetics , Protein Binding/genetics , Protein Binding/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolismABSTRACT
The 14-3-3 proteins comprise a family of highly conserved proteins with multiple functions, most of which are related to signal transduction. Four isoforms from the plant Arabidopsis thaliana were able to complement the lethal disruption of the two Saccharomyces cerevisiae genes encoding 14-3-3 proteins; one complemented very poorly and one did not complement. However, the expression of the latter two isoforms was very low. These results show that at least four of the six A. thaliana isoforms are able to perform the same function(s) as the yeast 14-3-3 proteins.
Subject(s)
Arabidopsis/physiology , Fungal Proteins/physiology , Proteins/physiology , Saccharomyces cerevisiae Proteins , Tyrosine 3-Monooxygenase , 14-3-3 Proteins , Arabidopsis/genetics , Base Sequence , Blotting, Western , DNA, Complementary , DNA, Fungal , Fungal Proteins/genetics , Genetic Complementation Test , Humans , Molecular Sequence Data , Proteins/geneticsABSTRACT
Seventy-six red adenine mutants of Kluyveromyces lactis were isolated. By complementation they could be assigned to two groups with 31 and 45 mutants. Transformation of several strains from each group with plasmids containing the Saccharomyces cerevisiae ADE1 or ADE2 gene showed that the largest group was ade2 and the other group was ade1. Several previously isolated 'ade1' mutants were classified to either group and given new gene and allele numbers. ADE1 was localized at chromosome III, closely linked to the mating type gene, making it a convenient marker for mating type. ADE2 was localized at chromosome V.
Subject(s)
Adenine/metabolism , Genes, Fungal , Genes, Mating Type, Fungal , Kluyveromyces/genetics , Mutation , Saccharomyces cerevisiae/genetics , Chromosome Mapping , Cloning, Molecular , Crosses, Genetic , Genetic Complementation Test , Genetic Linkage , Kluyveromyces/classification , Kluyveromyces/metabolism , Transformation, GeneticABSTRACT
The 2 microns plasmid of the yeast Saccharomyces cerevisiae is in principle a suitable vector for expression of foreign genes, due to its high copy number and extreme stability. However, the cloning of genes into 2 microns often results in a reduced copy number and/or reduced stability. One reason for this observed instability could be that the inserts in general were made in one of the several open reading frames (ORFs) of the plasmid. Therefore we studied the effect on stability of insertions in the silent regions of 2 microns without interrupting any known essential regions or ORFs. Using the SnaBI site, a yeast-integrating plasmid (Yip5) was introduced into the region between the ARS and STB locus in two possible orientations. The resulting plasmids could be stably maintained in the cells without the need for complementation by the wild-type 2 microns plasmid. However, the stability of these plasmids in a cir. host was still one to two orders of magnitude lower (0.2% and 0.8% respectively) as reported for the wild-type 2 microns (0.01%). Removal of 2 kb of the bacterial sequences from Yip5 did not increase stability. The stability was dependent on the orientation of the insert. We found that in the less stable orientation, transcription originating from the insert was running into the STB region. DNA inserted in the XmaIII site located outside the ORFs in the REP2/FLP intergenic region influenced both stability and copy number of the plasmid. These effects are strongly dependent on the size of the insert. Insertion of a 2 kb DNA fragment increased the copy number, probably through an effect on FLP expression.
Subject(s)
Mutagenesis, Insertional , Plasmids , Saccharomyces cerevisiae/genetics , Blotting, Northern , DNA, Fungal/chemistry , RNA, Fungal/analysis , Transcription, GeneticABSTRACT
A cDNA coding for the carboxy-terminal region of human protein S and containing a complete 3'-untranslated region, was isolated by a combination of antibody screening of a lambda gt11 human liver cDNA expression library and in situ hybridization of a pUC9 human liver cDNA library. Hybridization analysis of human total DNA with radiolabelled non-overlapping cDNA restriction fragments revealed the existence of two genes per haploid genome homologous with the protein S cDNA. Both genes were mapped to chromosome 3 using human-rodent cell hybrids. Neither of the genes showed polymorphism for sixteen different enzymes upon hybridization with the protein S cDNA.
Subject(s)
Chromosomes, Human, Pair 3 , DNA/genetics , Glycoproteins/genetics , Chromosome Mapping , Exons , Genes , Humans , Introns , Molecular Sequence Data , Polymorphism, Restriction Fragment Length , Protein S , Sequence Homology, Nucleic AcidABSTRACT
Partial cDNAs coding for human protein S were isolated from a pUC9 human liver cDNA library. Together, the overlapping clones span a (partial) 5'-non-coding region, and the complete protein S coding and 3'-untranslated regions. The derived amino acid sequence deviates at five positions from two previously reported protein S sequences. Two of these differences (Phe instead of Leu at position -16 and Tyr instead of Asp at position 222) are found in regions that are important for the post-translational modification of protein S, the gamma-carboxylation of glutamic acid and the hydroxylation of asparagine, respectively.