Expression of the genes for arylamine N-acetyltransferases in mice

Thesis

Arylamine N-acetyltransferases (NATs) are polymorphic enzymes involved in the metabolism of arylamine and hydrazine xenobiotics. Murine NAT isoenzymes are encoded by three Nat genes. Only Nat2 was previously known to be polymorphic, a single nucleotide substitution causing the slow acetylator phenotype in the A/J strain. The present study (Chapter 3) describes novel polymorphisms in all three Nat genes of the wild-derived inbred strains Mus spretus and Mus musculus castaneus. Functional analysis of hepatic and heterologously expressed NAT variants from the two strains demonstrated that M. m. castaneus is a fast and M. spretus a slow acetylator.

A previously isolated 14.3kb Nat-positive mouse genomic clone of 129/Ola strain origin (clone A) was sequenced (Chapter 4). A 8.6kb HindIII fragment, containing the entire Nat2 coding region, was subcloned from clone A and used to generate a targeting construct for the production of Nat2 knock-out mice. Probes and appropriate PCR methodologies were developed for screening of embryonic stem cells for targeted incorporation of the construct (Chapter 4). Computational analysis of clone A sequence revealed a number of important elements around the Nat2 gene, including four microsatellite markers which were found to be polymorphic among different mouse strains. Combined with preliminary physical mapping work (Chapter 4), these markers will assist accurate localisation of the Nat genes on mouse chromosome 8.

A non-coding exon was mapped 6.4-6.1kb upstream of the intronless Nat2 coding region. A functional polyadenylation signal was also identified 0.45kb downstream of the mouse Nat2 coding region. The genomic structure of other genes for mammalian NAT was also analysed, by comparison of ESTs and genomic sequences deposited in electronic databases. Reverse transcription PCR confirmed that Nat2 is expressed in many tissues, while Nat1 and Nat3 are expressed in the liver and spleen, respectively. The Nat2 transcript was also detected in mouse embryonic stem cells, suggesting a possible involvement of murine NAT2 early in development (Chapter 5). The elements constituting the core promoter of Nat2 were characterised, and a preliminary search for other transcriptional regulatory sequences was carried out, using reporter gene assays and electrophoretic mobility shift assays (Chapter 6).

Authors: Boukouvala, S; Boukouvala, Sotiria

• Publication date: 2002
• DOI: 10.5287/ora-pyjpqknjx
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Subjects: Pharmacogenetics; Gene Mapping