ISOLATION AND CHARACTERIZATION OF CDNA CLONES ENCODING PIG GASTRIC MUCIN

Polyclonal antibodies raised to deglycosylated pig gastric mucin were used to screen a cDNA library constructed with pig stomach mucosal mRN A. Immunocytochemistry indicated that the antibody recognizes intracel lular and secreted mucin in surface mucous cells of pig gastric epithe lium. A total of 70 clones producing proteins immunoreactive to this a ntibody were identified, two of which (PGM-2A,9B) were fully sequenced from both ends, Clone PGM-9B hybridized to a polydisperse mRNA (3-9 k b) from pig stomach, but not liver, intestine or spleen, nor to mRNA f rom human, mouse, rabbit or rat stomach. Sequence analysis indicated t hat PGM-9B encodes 33 tandem repeats of a 16-amino-acid consensus sequ ence rich in serine (46%) and threonine (17%). Using the restriction e nzyme MwoI, which has a single target site in the repeat, it was demon strated that PGM-9B consists entirely of this tandem repeat. Southern- blot analysis indicated that the repeat region is contained in a 20 kb HindIII-EcoRI fragment, and BamHI digestion suggested that most of th e repeats are contained in a 10 kb fragment. In situ hybridization wit h an antisense probe to PGM-9B showed an intense signal in the entire gastric gland. Clone PGM-2A also contains the same repeat sequence as 9B, but, in addition, has a 64-amino-acid-long non-repeat region at it s 5' end. Interestingly the nonrepeat region of PGM-2A has five cystei ne residues, the arrangement of which is identical with that reported for human intestinal mucin gene MUC2.