USE OF ALTERNATE SPLICE SITES IN GRANULE-BOUND STARCH SYNTHASE MESSENGER-RNA FROM LOW-AMYLOSE RICE VARIETIES

The rice Waxy gene encodes a granule-bound starch synthase (GBSS) nece ssary for the synthesis of amylose in endosperm tissue. We have previo usly shown that a CT microsatellite near the transcriptional start sit e of the GBSS gene can distinguish 7 alleles that accounted for more t han 80% of the Variation in apparent amylose content in an extended pe digree of 89 US rice cultivars (Oryza sativa L.). Furthermore, all the cultivars with 18% or less amylose were shown to have the sequence AG (T) under bar TATA at the putative leader intron 5' splice site, whil e all cultivars with a higher proportion of amylose had AG (G) under b ar TATA. Here we demonstrate that this single-base mutation reduces th e efficiency of GBSS pre-mRNA processing and results in alternate spli cing at three cryptic sites. The predominant 5' splice site in CT18 lo w-amylose varieties is 93 bp upstream of the splice site used in inter mediate and high amylose varieties and is immediately 5' to the CT mic rosatellite that we previously demonstrated to be tightly correlated w ith amylose content. Use of the leader intron 5' splice site at either -93 or -1 in conjunction with the predominant 3' splice site results in formation of a small open reading frame 38 bp upstream of the norma l ATG and out of frame with it. This open reading frame is not produce d when any of the 5' leader intron splice sites are used in conjunctio n with an alternate 3' splice site five bases further downstream which was observed in all rice varieties tested.