CLONING AND SEQUENCING OF A FULL-LENGTH RAT SUCRASE-ISOMALTASE-ENCODING CDNA

Sucrase-isomaltase (SI) has been widely used as a marker enzyme to stu dy cellular differentiation in the small intestine. We isolated a 6.1- kb SI cDNA clone (GC1,4) from a size-fractionated cDNA library from ra t intestine. Sequencing of this cDNA clone showed 6066 nucleotides (nt ) with an open reading frame (ORF) of 1841 amino acids (aa). The nt se quence correctly predicts several known aa stretches in the protein. T he deduced aa sequence showed 78 and 75% overall identity with the rab bit and human SI, respectively. At the active sites of both S and I, t he rat nt sequence encodes stretches of 14 and 16 aa, respectively, wh ich show 100% identity to rabbit and human SI. In the region immediate ly beyond the transmembrane domain, the rat sequence encodes an extra 10 aa, as compared to rabbit and human. This 10-aa insertion consists almost entirely of Pro, Ser and Thr, and may be responsible for additi onal O-glycosylations of rat SI. The cDNA contains a 3'-UTR (untransla ted region) of 499 nt with polyadenylation signal sequence and a poly( A) tract. The ATG start codon was found 41 nt downstream from the 5' e nd of the cDNA. Primer extension experiments showed the cap site to be 61 nt upstream from the start codon, The results indicate that our cD NA clone lacks only 20 nt in the 5'-UTR. Given that this cDNA encodes the entire coding region of SI, it should be useful in elucidating the regulatory mechanisms of SI biosynthesis, localization and targeting during rat intestinal development and differentiation.