Expression of the genes for arylamine N-acetyltransferases in mice
<p xmlns:etd="http://www.ouls.ox.ac.uk/ora/modsextensions">Arylamine N-acetyltransferases (NATs) are polymorphic enzymes involved in the metabolism of arylamine and hydrazine xenobiotics. Murine NAT isoenzymes are encoded by three <em>Nat</em> genes. Only <em>Nat2&l...
| Main Authors: | , |
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| Other Authors: | |
| Format: | Thesis |
| Language: | English |
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2002
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| _version_ | 1826303588699209728 |
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| author | Boukouvala, S Boukouvala, Sotiria |
| author2 | Sim, E |
| author_facet | Sim, E Boukouvala, S Boukouvala, Sotiria |
| author_sort | Boukouvala, S |
| collection | OXFORD |
| description | <p xmlns:etd="http://www.ouls.ox.ac.uk/ora/modsextensions">Arylamine N-acetyltransferases (NATs) are polymorphic enzymes involved in the metabolism of arylamine and hydrazine xenobiotics. Murine NAT isoenzymes are encoded by three <em>Nat</em> genes. Only <em>Nat2</em> 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 <em>Nat</em> genes of the wild-derived inbred strains <em>Mus spretus</em> and <em>Mus musculus castaneus</em>. Functional analysis of hepatic and heterologously expressed NAT variants from the two strains demonstrated that <em>M. m. castaneus</em> is a fast and <em>M. spretus</em> a slow acetylator.</p> <p xmlns:etd="http://www.ouls.ox.ac.uk/ora/modsextensions">A previously isolated 14.3kb <em>Nat</em>-positive mouse genomic clone of 129/Ola strain origin (clone A) was sequenced (Chapter 4). A 8.6kb <em>Hind</em>III fragment, containing the entire <em>Nat2</em> coding region, was subcloned from clone A and used to generate a targeting construct for the production of <em>Nat2</em> 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 <em>Nat2</em> 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 <em>Nat</em> genes on mouse chromosome 8.</p> <p xmlns:etd="http://www.ouls.ox.ac.uk/ora/modsextensions">A non-coding exon was mapped 6.4-6.1kb upstream of the intronless <em>Nat2</em> coding region. A functional polyadenylation signal was also identified 0.45kb downstream of the mouse <em>Nat2</em> 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 <em>Nat2</em> is expressed in many tissues, while <em>Nat1</em> and <em>Nat3</em> are expressed in the liver and spleen, respectively. The <em>Nat2</em> 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 <em>Nat2</em> 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).</p> |
| first_indexed | 2024-03-07T06:04:59Z |
| format | Thesis |
| id | oxford-uuid:ed864a10-2cb9-4ebe-8a2c-934d707c5a0d |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T06:04:59Z |
| publishDate | 2002 |
| record_format | dspace |
| spelling | oxford-uuid:ed864a10-2cb9-4ebe-8a2c-934d707c5a0d2022-03-27T11:25:42ZExpression of the genes for arylamine N-acetyltransferases in miceThesishttp://purl.org/coar/resource_type/c_db06uuid:ed864a10-2cb9-4ebe-8a2c-934d707c5a0dGene MappingPharmacogeneticsEnglishPolonsky Theses Digitisation Project2002Boukouvala, SBoukouvala, SotiriaSim, ESim, E<p xmlns:etd="http://www.ouls.ox.ac.uk/ora/modsextensions">Arylamine N-acetyltransferases (NATs) are polymorphic enzymes involved in the metabolism of arylamine and hydrazine xenobiotics. Murine NAT isoenzymes are encoded by three <em>Nat</em> genes. Only <em>Nat2</em> 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 <em>Nat</em> genes of the wild-derived inbred strains <em>Mus spretus</em> and <em>Mus musculus castaneus</em>. Functional analysis of hepatic and heterologously expressed NAT variants from the two strains demonstrated that <em>M. m. castaneus</em> is a fast and <em>M. spretus</em> a slow acetylator.</p> <p xmlns:etd="http://www.ouls.ox.ac.uk/ora/modsextensions">A previously isolated 14.3kb <em>Nat</em>-positive mouse genomic clone of 129/Ola strain origin (clone A) was sequenced (Chapter 4). A 8.6kb <em>Hind</em>III fragment, containing the entire <em>Nat2</em> coding region, was subcloned from clone A and used to generate a targeting construct for the production of <em>Nat2</em> 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 <em>Nat2</em> 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 <em>Nat</em> genes on mouse chromosome 8.</p> <p xmlns:etd="http://www.ouls.ox.ac.uk/ora/modsextensions">A non-coding exon was mapped 6.4-6.1kb upstream of the intronless <em>Nat2</em> coding region. A functional polyadenylation signal was also identified 0.45kb downstream of the mouse <em>Nat2</em> 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 <em>Nat2</em> is expressed in many tissues, while <em>Nat1</em> and <em>Nat3</em> are expressed in the liver and spleen, respectively. The <em>Nat2</em> 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 <em>Nat2</em> 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).</p> |
| spellingShingle | Gene Mapping Pharmacogenetics Boukouvala, S Boukouvala, Sotiria Expression of the genes for arylamine N-acetyltransferases in mice |
| title | Expression of the genes for arylamine N-acetyltransferases in mice |
| title_full | Expression of the genes for arylamine N-acetyltransferases in mice |
| title_fullStr | Expression of the genes for arylamine N-acetyltransferases in mice |
| title_full_unstemmed | Expression of the genes for arylamine N-acetyltransferases in mice |
| title_short | Expression of the genes for arylamine N-acetyltransferases in mice |
| title_sort | expression of the genes for arylamine n acetyltransferases in mice |
| topic | Gene Mapping Pharmacogenetics |
| work_keys_str_mv | AT boukouvalas expressionofthegenesforarylaminenacetyltransferasesinmice AT boukouvalasotiria expressionofthegenesforarylaminenacetyltransferasesinmice |