Title

A molecular genetic and cellular characterization of the acetaminophen and selenium binding proteins

Date of Completion

January 1996

Keywords

Biology, Molecular|Biology, Genetics|Biology, Cell

Degree

Ph.D.

Abstract

Liver cytotoxicity after acetaminophen (APAP, N-acetyl p-aminophenol) overdose has been associated with binding of the reactive metabolite, N-acetylbenzoquinoneimine (NAPQI), to cellular proteins. Because APAP toxicity is correlated with covalent binding, protein binding is believed to play a role in cell death. The major cytosolic acetaminophen binding protein of 56/58 kDa (58-ABP) has been purified.^ Several peptides of the 58-ABP have been sequenced and shown to be homologous to two cDNAs: ABP and SBP. These cDNAs are 97% identical at the nucleotide level with only 20 nucleotide differences dispersed throughout the protein coding region. The mRNA of the SBP and the 58-ABP are both found in mouse liver, lung, and kidney which are all tissues where acetaminophen binds to proteins and causes toxicity. In contrast, ABP mRNA is found only in liver. Little is known about the function of the SBP and ABP; but based on the high degree of homology between the two cDNA's, they may serve related cellular functions.^ In order to characterize these two proteins at the nucleic acid level we have developed an assay to distinguish between these two proteins at the genomic DNA level. Using this assay we have shown that these two proteins are indeed coded for by two different genes and not alternatively spliced transcripts and have also identified a third gene (gene X). Using this assay we cloned the genomic DNA for both genes. We have used these genomic clones to sequence the 5$\sp\prime$UTR of the genes and found a unique gene structure within this region for both genes. Sequencing further upstream of the ABP gene we have found many putative transcription factor binding sites.^ Further analysis of these genes led us to map the location of the ABP and SBP within the mouse genome. We have found that the two genes show tight linkage on mouse chromosome 3, with gene X mapping to chromosome 4. These results indicate that the ABP and SBP are closely related and may represent a gene family. ^

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