Cloning of the Amino-terminal and 5′-Flanking Region of the Human MUC5AC Mucin Gene and Transcriptional Up-regulation by Bacterial Exoproducts

Abstract

To obtain gene regulatory sequence for the mucin gene MUC5AC, we have isolated the MUC5AC amino terminus cDNA and 5'-flanking region. This was possible through the use of rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) in which the 5' sequence of the human gastric mucin cDNA HGM-1 (1) was used to design the first MUC5AC-specific primer. Primers for subsequent rounds of RACE were designed from the 5'-ends of amplified RACE products. After five rounds of RACE-PCR, we could no longer generate upstream extensions of the cDNA and hypothesized that we had reached the 5'-end. Primer extension and RNase protection analysis confirmed this. Combined nucleotide sequence for the RACE-PCR products was 3.3 kb with an open reading frame encoding 1100 amino acids. A putative translation start site was found at nucleotide +48. This was followed by a 45 nucleotide putative signal sequence. This amino-terminal sequence contains no tandem repeats but is >60% similar to the amino-terminal nucleotide sequence of MUC2. The positions of cysteine residues in this MUC2-similar region are almost 100% conserved between the two genes. Northern analysis showed expression of cognate RNA in the stomach and airway but not muscle and esophagus. This pattern was the same as that obtained using previously reported 3'-MUC5AC sequences. We have cloned approximately 4 kb of genomic DNA upstream of the transcription start site and have sequenced 1366 nucleotides containing a TATA box, a CACCC box, and putative binding sites for NFkappaB and Sp 1. Within 4 kb of the transcription start site are elements mediating transcriptional up-regulation in response to bacterial exoproducts.