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Identification of N-acetyltransferase 2 (NAT2) transcription start sites and quantitation of NAT2-specific mRNA in human tissues.

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.

SUBMITTER: Husain A

PROVIDER: S-EPMC1931608 | biostudies-literature | 2007 May

REPOSITORIES: biostudies-literature

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Identification of N-acetyltransferase 2 (NAT2) transcription start sites and quantitation of NAT2-specific mRNA in human tissues.

Husain Anwar A Zhang Xiaoyan X Doll Mark A MA States J Christopher JC Barker David F DF Hein David W DW

Drug metabolism and disposition: the biological fate of chemicals 20070207 5

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 ...[more]

PMID: 17287389

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Project description:Despite almost 40 years of molecular genetics research in Escherichia coli a major fraction of its Transcription Start Sites (TSSs) are still unknown, limiting therefore our understanding of the regulatory circuits that control gene expression in this model organism. RegulonDB (http://regulondb.ccg.unam.mx/) is aimed at integrating the genetic regulatory network of E. coli K12 as an entirely bioinformatic project up till now. In this work, we extended its aims by generating experimental data at a genome scale on TSSs, promoters and regulatory regions. We implemented a modified 5' RACE protocol and an unbiased High Throughput Pyrosequencing Strategy (HTPS) that allowed us to map more than 1700 TSSs with high precision. From this collection, about 230 corresponded to previously reported TSSs, which helped us to benchmark both our methodologies and the accuracy of the previous mapping experiments. The other ca 1500 TSSs mapped belong to about 1000 different genes, many of them with no assigned function. We identified promoter sequences and type of sigma factors that control the expression of about 80% of these genes. As expected, the housekeeping sigma(70) was the most common type of promoter, followed by sigma(38). The majority of the putative TSSs were located between 20 to 40 nucleotides from the translational start site. Putative regulatory binding sites for transcription factors were detected upstream of many TSSs. For a few transcripts, riboswitches and small RNAs were found. Several genes also had additional TSSs within the coding region. Unexpectedly, the HTPS experiments revealed extensive antisense transcription, probably for regulatory functions. The new information in RegulonDB, now with more than 2400 experimentally determined TSSs, strengthens the accuracy of promoter prediction, operon structure, and regulatory networks and provides valuable new information that will facilitate the understanding from a global perspective the complex and intricate regulatory network that operates in E. coli.

Dataset Information

Identification of N-acetyltransferase 2 (NAT2) transcription start sites and quantitation of NAT2-specific mRNA in human tissues.

Publications

Identification of N-acetyltransferase 2 (NAT2) transcription start sites and quantitation of NAT2-specific mRNA in human tissues.

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