Christine E. Seyfried

Purification, properties and regulation of the level of bovine S-adenosylmethionine decarboxylase during lymphocyte mitogenesis

S-adenosylmethionine decarboxylase was purified from the livers of calves treated with methylglyoxal bis(guanylhydrazone) to elevate the level of the enzyme. Purified bovine S-adenosylmethionine decarboxylase was similar in specific activity and subunit molecular weight (32,000) to the enzymes previously isolated from rat and mouse. The bovine liver enzyme immunologically crossreacted with S-adenosylmethionine decarboxylase from resting and mitogenically activated bovine lymphocytes. The rate of enzyme synthesis in activated lymphocytes was determined by labeling the cells with [3H]leucine and isolating the radioactive decarboxylase by affinity chromatography and sodium dodecyl sulfate gel electrophoresis. The rate of enzyme synthesis was increased 10-fold by 9 h after mitogen treatment, which accounts for the initial increase in cellular enzymatic activity. There was no further increase in the rate of S-adenosylmethionine decarboxylase synthesis that correlated with a second elevation of activity occurring at approx. 24 h after mitogenic activation. It was concluded that the second increase in enzyme activity was due to lengthening the intracellular half-life of the enzyme by 2-fold.

Sequence analysis of HLA class II domains: characterization of the DQw3 family of DQB genes

HLA class II allelic variants within the DQw3-related family of genes carry distinct allo-specificities and have been implicated in specific HLA-disease associations, such as insulin-dependent diabetes mellitus. To investigate the nucleotide variations which characterize DQw3 genes, we applied a novel cDNA cloning strategy that uses a single-stranded vector/primer system to facilitate DNA sequencing of allelically variable gene families. Using a DQB-specific primer sequence and M13 bacteriophage as the cloning vector, direct cloning and sequencing of multiple DQB genes was performed without the need for second strand synthesis or for subcloning. Sequence analysis from eight lymphoblastoid cell lines selected to represent different ethnic backgrounds revealed three DQw3-related DQB genes, DQB3.1, 3.2, and 3.3, corresponding to the newly designated HLA-DQw7, w8, and w9 specificities, respectively. An unusual Pro-Pro couplet at codons 55–56 is characteristic of all DQw3-positive sequences and may be contributing to the broad DQw3 allospecificity. Comparisons among ethnically disparate DQw3-related sequences showed no additional expressed or silent nucleotide substitutions among these DQB alleles. Thus, polymorphism within the DQw3 family of genes appears to be extremely limited, with a paucity of nucleotide variations accumulated by evolutionary distance.

Functional polymorphisms among HLA-DR4+ DR beta chains associated with limited peptide diversity☆

Tryptic peptide map analysis by high-pressure liquid chromatography of DR4- associated DR beta chains revealed limited structural variation within DR beta polypeptides. Comparison of 3H-leucine-labelled tryptic peptide maps of Dw4 and Dw14 homozygous cells identified distinct peaks corresponding to Dw4 and Dw14-associated DR beta polypeptides. HPLC analysis of cell line 256, heterozygous for two DR4-related specificities, Dw4 and Dw14, displayed both peptides, corresponding to the variable Dw4 and Dw14 chromatograms. This observation was confirmed using a deletion mutant cell line derived from 256 lacking Dw4-associated class II genes. The observed peptide variation correlated precisely with predicted nucleotide-derived amino acid sequences implicating amino acids 66-71 of the DR beta chain as contributing to HLA-D structural and functional polymorphisms.

HLA-DR4-Associated Disease: Oligonucleotide Probes Identify Specific Class II Susceptibility Genes in Type I Diabetes and Rheumatoid Arthritis

Distinct patterns of linked DRβ (HLA-DRB1) and DQβ (HLA-DQB1) genes are found on at least seven different haplotypes that carry the HLA-DR4 specificity (1,2). We have constructed 20 and 21-base synthetic oligonucleotide DNA probes capable of distinguishing between different DRβ alleles and between different DQβ alleles expressed on these DR4-positive haplotypes. Hybridization analysis indicates striking differences in the distribution of individual DQβ and DRβ alleles in DR4-positive patients with rheumatoid arthritis (RA) and DR4-positive patients with insulin-dependent diabetes mellitus (IDDM, type I diabetes). Probes identifying Dw14 or Dw4 DRβ genes hybridized to greater than 93% of patients with seropositive rheumatoid arthritis, consistent with the concept that these two alleles account for the DR4 association with this disease. No difference was observed between the distribution of different DQβ alleles in rheumatoid arthritis as compared to normal individuals. In contrast, DQβ 3.2 specific oligonucleotide probes hybridized to greater than 96% of DR4-positive IDDM patients, regardless of the linked DR4-positive DRβ gene.