Identification ofN-Acetyltransferase 2 (NAT2) Transcription Start Sites and Quantitation ofNAT2-Specific mRNA in Human Tissues

Abstract

ABSTRACT:Human N-acetyltransferase 2 (NAT2) genetic polymorphism is associated with drug toxicity and/or carcinogenesis in various tissues. Knowledge of NAT2 gene structure and expression is critical for understanding these associations. Previous findings suggest that human NAT2 expression is highest in liver and gut but expressed at functional levels in other tissues. A sensitive and specific TaqMan reverse transcriptase-polymerase chain reaction (RT-PCR) assay with intron-spanning primers was developed and used, together with a second TaqMan RT-PCR assay based on amplification of a NAT2 open reading frame (ORF) exon segment, to measure NAT2 mRNA in 29 different human tissues. Cap-dependent amplification of mRNA 5 termini and review of public database information were done to more precisely define the NAT2 promoter(s) and to validate the quantitative RT-PCR assay design. The great majority (40/41) of NAT2 liver cDNAs had 5 termini between 8682 and 8752 nucleotides upstream of the NAT2 ORF exon, and 34 of 40 5 termini were at the ؊8711 and ؊8716 adenines. All 59 NAT2 cDNAs with 5 termini in this vicinity, including 40 of the liver isolates and 19 cDNAs in public databases from liver and other sources, showed direct splicing to the ORF exon, with no other noncoding exon detected. NAT2 mRNA was highest in liver, small intestine, and colon and was readily detected in most other tissues, albeit at much lower levels. NAT2 expression in diverse human tissues provides further mechanistic support underlying associations between NAT2 genetic polymorphism, drug toxicity, and/or chemical carcinogenesis.Genetic polymorphism of the N-acetyltransferase 2 gene (NAT2) is strongly implicated in differential susceptibility to adverse drug reactions (Weber and Hein, 1985;Butcher et al., 2002) and to various diseases (Boukouvala and Fakis, 2005), especially cancers of the urinary bladder (Garcia-Closas et al., 2005;Carreon et al., 2006;Hein, 2006) and colon (Lilla et al., 2006;Moslehi et al., 2006), on exposure to carbocyclic and heterocyclic-aromatic amine carcinogens. A large number of studies have also examined the possible involvement of NAT2 in the etiology of cancers of various other organs (reviewed in Hein et al., 2000a,b). A critical question is whether vulnerability to neoplastic transformation is related to specific expression of NAT2 in the target organ (Williams, 2001), and studies have investigated NAT2 expression in various human (reviewed in Boukouvala and Fakis, 2005) and recently in Syrian hamster and mouse (Loehle et al., 2006) tissues. We hypothesized that human NAT2 expression is highest in liver and gut but is also present in other human tissues. To test this hypothesis, sensitive, specific, and well characterized quantitative mRNA assays were used to quantitate relative expression of NAT2 in 29 different human tissue types.Accurate quantitative measurements of NAT2 mRNA and protein pose a particular technical challenge because of the presence of the paralogous gene, N-acetyltransferase 1 (NAT1), which is ubi...

Cloning and Characterization of the Rat Lysyl Oxidase Gene Promoter

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“…2B) (30), which has proven to be a very sensitive and accurate method to identify full-length 5Ј ends of cDNAs by eliminating truncated messages from the amplification reactions. The advantage of this method over others is that only authentic capped 5Ј ends of mRNA are detected.…”

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Synergism between the N-acetyltransferase 2 gene and oxidant exposure increases the risk of idiopathic male infertility

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