Identification of the major promoter and non-coding exons of the human arylamine N-acetyltransferase 1 gene (NAT1)

Abstract

Some carcinogens that initiate rat mammary cancer are substrates of human N-acetyltransferase 1 (NAT1) and variation in NAT1 activity due to environmental or genetic causes may influence human susceptibility to breast cancer. One unexplored potential cause of NAT1 expression variation is polymorphism of transcriptional control sequences. However, the location of the major NAT1 transcription control site is uncertain because earlier publications and current databases report different cDNA structures. To resolve this discrepancy, we used CAP-dependent cDNA cloning to identify 5' ends of NAT1 mRNAs from breast and MCF-7, a mammary adenocarcinoma cell line. Most transcription initiates in a 49-bp region located 11.8 kb upstream of the coding exon. A 79-bp exon located 2.5 kb upstream of the coding exon was found in all 41 of the independent NAT1 cDNA products. Seven of these 41 cDNAs also included other non-coding exons. The structures of NAT1 cDNAs in public databases, as obtained from diverse tissues, reflect a transcription pattern similar to that demonstrated in breast and MCF-7. Genomic fragments spanning the major start region were cloned into a luciferase vector and expressed in MCF-7. Promoter activities were 190-490-fold higher than the vector control and 30-80-fold higher than for a fragment immediately upstream of the coding exon. Our results demonstrate that, in breast, and likely also in other tissues, the major NAT1 mRNA is transcribed from a strong promoter located 11.8 kb upstream of the translated exon, and the mature spliced mRNA includes at least one additional non-coding exon.

Functional effects of single nucleotide polymorphisms in the coding region of human N-acetyltransferase 1

“…Many of the NAT1 haplotypes that have been identified in human populations contain a single SNP in the 870 bp NAT1 coding region (Table 1). Although the functional effects of SNPs in the promoter and other regions of the NAT1 gene may also affect phenotype, 22,23 our investigation focused on the functional effects of the SNPs identified in the NAT1 coding region.…”

Bioactivation, protein haptenation, and toxicity of sulfamethoxazole and dapsone in normal human dermal fibroblasts☆

“…NAT1-specific mRNA levels were determined as previously described (Husain et al, 2004). NAT2-specific mRNA levels were determined in an identical manner except that the forward NAT2 5′-ttggaagcaagaggattgcat-3′ and reverse NAT2 5′-gatctggtgctcaagaatgtcagt-3′ primers spanned an 8.6 kb intron in NAT2 and the TaqMan probe was fam-tcctggttgctggcc.…”

“…Absolute levels of NAT1 and NAT2 mRNA were determined by calibration to a standard curve prepared with plasmids containing amplified NAT1 and NAT2 mRNA standards quantified by UV absorbance at 260 nm. All samples and standards were normalized to beta-actin mRNA as described previously (Husain et al, 2004).…”

Functional analysis of N-acetyltransferase (NAT1*14B and NAT1*10) in complete NATb and NATa mRNA.

“…The NA T1 gene is approximately 53 kb in length and contains at least nine exons (Husain et aL, 2004). Many NAT1 transcripts have been identified containing different combinations of the 5' UTR exons originating from two separate promoters (Husain et aL, 2004;Butcher et aL, 2005). Differences in translational efficiencies exist between transcripts originating at each promoter, but the biological importance of this remains unclear (Butcher et aL, 2005).…”

“…The NA T1 gene is approximately 53 kb in length and contains at least nine exons (Husain et aL, 2004). Many NAT1 transcripts have been identified containing different combinations of the 5' UTR exons originating from two separate promoters (Husain et aL, 2004;Butcher et aL, 2005).…”