The Basidiomycete ras gene possesses a pyrimidine-rich stretch (CT-motif) with a short (7 bases) mirror repeat in which its major transcription start point is contained, To analyze the tertiary structure induced by the CT/AG-biased sequence and its effect on gene expression in supercolied plasmids in Escherichia coli, the DNA fragment containing the ras CT/AG sequence was inserted into the EcoRI site on pBR322 in both orientations and the resulting pBR322 derivatives, named pBR-CT[ras] and pBR-invCT[ras] were introduced into E. coli strains DM800 (Delta topA gyrB225) and JM109 (topA(+) gyrA96), In pBR-CT [ras] the pyrimidine-rich sequence is on the pBR322 tetracycline-resistance gene (tet)-coding strand and in pBR-invCT[ras] the complementary purine-rich sequence is on this strand, DNAs of pBR-CT[ras] and pBR-invCT[ras] isolated from DM800 were frequently cleaved with single-strand-specific S1 nuclease within the CT/AG sequence, showing the formation of extended open structure, Compared with those carrying pBR322, DM800 and JM109 carrying pBR-CT[ras] showed much higher levels of tetracycline resistance (Tc-r), while both strains carrying pBR-invCT[ras] showed clearly lower levels of Tc-r, pBR-CT [ras] and pBR-invCT[ras], however, conferred reduced activity of beta-lactamase on DM800 and JM109. pBR-CT[ras] derivatives lacking the counterpart of the mirror repeat did not form the S1-cleavable open structure within the CT/AG sequence and conferred pBR322-like Tc-r and beta-lactamase activity, The tertiary structure formed in the CT/AG sequence via the mirror repeat was suggested to affect the expressions of pBR322-tet and -bla genes.