The autonomous replication function (ARS) of the scaffold-associated region (SAR) of silkworm attacus ricini rDNA in yeast

The one kilo-base scaffold-associated region (SAR) of silkworm Attacus ricini rRNA gene (rDNA) has been identified previously^[1]. To investigate the critical sequence and the relative activity of ARS (autonomously replicating sequence), a set of restriction from covered the whole rRNA gene unit were subcloned into the nonreplicative pSKY vector. Among the seven plasmids constructed, the plasmid pSEY, having SAR, gave obvious positive replication activity in yeast as determined by the transformation efficiency. Further dissection of SAR demonstrated that plasmid pAAY, having a 0.26 kb small fragment of SAR was 40-50 fold more active then the whole SAR, while pSAY, having the remaining part of SAR, showed no activity. There were fifteen ARS consensus sequence (ACS) within SAR being identified through sequence alignment. It was found that only three ACSs in pAAY, located at the 3′ end of SAR displayed a positive ARS activity and the remaining sequence having the other twelve ACSs functioned as a repressor. The in vitro binding assay showed that SAR from the silkworm rRNA gene bound to the yeast nuclear scaffold. These results suggest that SAR is evolutionarily conserved, and there is a close correlation between SAR and ARS activity. Detailed analysis of the positive and negative regulatory elements in SAR can be carried out based on these results.