Molecular cloning and preliminary analysis of a fragile site associated gene.

OBJECTIVE To analyze the molecular coining of a fragile site-associated gene. METHODS Genomic Chinese hamster ovary (CHO) DNA library was constructed using high molecular weight CHO DNA partially digested with MboI restriction enzyme from cultured CHO cells. Screening of genomic DNA library followed the established procedures. Genomic CHO in the positive clones was sequenced. Appropriate primers were designed for the reverse transcriptase-polymerase chain reactions (RT-PCR). The RT-PCR products were cloned into a pCRII TOPO vector and confirmed by DNA sequencing. Antibodies were prepared using synthetic peptides as antigens by immunizing the rabbits. Immunohistochemical analyses were performed to evaluate the expression of the novel gene in different tissues. RESULTS To investigate the molecular mechanism underlying the initial events of mdr1a amplification, we cloned 1q31 fragile site DNA. Strikingly, we found that this fragile site contained a novel gene which was designated as a fragile site-associated (FSA) gene. FSA encoded an unusually large mRNA of approximately16 kb. Full-length human FSA cDNA was cloned. FSA mRNA was expressed in many cultured cells and tissue types. Immunohistochemical analyses also revealed an expression pattern of the encoded proteins in postmitotic, well-differentiated epithelial compartments of many organs, including colon, mammary glands, ovary, prostate, and bladder. CONCLUSION FSA plays an important role in regulating mammalian epithelial cell growth and differentiation.