ABSTRACT
C282Y and H63D are two common variants of the hemochromatosis protein HFE. SH-SY5Y human neuroblastoma cells stably transfected to express either wild type HFE (WT-HFE), or the C282Y or H63D allele were analyzed for effect of expression of the mutant proteins on transcription of 14 enzymes involved in sphingolipid metabolism. Cells expressing the C282Y variant showed significant increases (>2-fold) in transcription of five genes and decreases in two compared to that seen for cells expressing WT-HFE, while cells expressing the H63D variant showed an elevation in transcription of one gene and a decrease in two. These changes were seen as alterations in ganglioside composition, cell surface binding by the binding subunit of cholera toxin, expression of sphingosine-kinase-1 and synthesis of sphingosine-1-phosphate. These changes may explain why C282Y-HFE is a risk factor for colon and breast cancer and possibly protective against Alzheimer's disease while H63D-HFE is a risk factor for neurodegenerative diseases.
Subject(s)
Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Neuroblastoma/metabolism , Point Mutation , Sphingolipids/metabolism , Alleles , Animals , Carbohydrate Conformation , Carbohydrate Sequence , Cell Line, Tumor , Gene Expression Regulation, Enzymologic , Genetic Predisposition to Disease , Genotype , Hemochromatosis Protein , Humans , Membrane Microdomains/chemistry , Molecular Sequence DataABSTRACT
Properties of the change from asparagine dependence (asn-) to independence (asn+) were investigated in the androgenetic haploid frog cell line ICR 2A. Two types of asn+ variants arose spontaneously during culture. Glutamine-dependent asparagine synthetase (AS) activity, found to be deficient in asn- cells, was repressed by asparagine in one type of variant and expressed constitutively in the other. No quantitative differences in AS-specific DNA sequences or changes in ploidy were evident between asn+ and asn- cells. The asn+ frequency in ICR 2A populations, not dramatically influenced by chemical mutagens, was increased 130-fold by exposure to 5-azacytidine. The methylation of CCGG sequences at the 5' end of the AS structural gene was found to be reduced equally in both types of asn+ variant. These results indicate that decreased DNA methylation is essential but not necessarily sufficient for the expression of AS activity in this frog cell system.
Subject(s)
Asparagine/metabolism , Genetic Variation , Haploidy , Animals , Aspartate-Ammonia Ligase/genetics , Aspartate-Ammonia Ligase/metabolism , Cell Line , DNA/analysis , DNA/metabolism , Karyotyping , Methylation , Mutagens/pharmacology , RNA, Messenger/analysis , Rana pipiens/embryologyABSTRACT
Nuclear poly(A) polymerase was isolated from [35S]methionine-labeled hepatoma McA-RH 7777 cells and subjected to DEAE-Sephadex chromatography. Flow-through and low salt wash fractions containing poly(A) polymerase activity were pooled and subjected to immunoblot analysis using anti-tumor type poly(A) polymerase antibodies and a biotinylated second antibody. The immune complex contained a single 48-kDa polypeptide band corresponding to the tumor-type enzyme. When immunoprecipitations were carried out using the same fraction and antibodies, at least five 35S-methionine-labeled proteins with approximate molecular masses of 74, 48, 35, 30, and 22 kDa were observed. Pulse-chase studies did not indicate a precursor-product relationship between the immunoprecipitated proteins. Preimmune sera did not react with poly(A) polymerase or other components in the protein complex. These data show that poly(A) polymerase exists as part of a complex with at least four other polypeptides and suggest that these polypeptides may be involved in the cleavage and/or polyadenylation reactions.
