Structure and function in Escherichia coli of plasmids containing pyrimidine/purine-biased stretch originated from the 5'-flanking region of the basidiomycete ras gene.
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 supercoiled 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 (deltatopA 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 (Tcr), while both strains carrying pBR-invCT[ras] showed clearly lower levels of Tcr. 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 Tcr 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.
Yamazaki
T
T
Department of Life Science, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama.
Hasebe
T
T
Shouguchi
J
J
Amano
H
H
Kajiwara
S
S
Shishido
K
K
D001483:Base Sequence; D001487:Basidiomycota / Q000235:genetics*; D004274:DNA, Recombinant / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005800:Genes, Fungal; D011905:Genes, ras; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010957:Plasmids; D011687:Purines / Q000378:metabolism*; D011743:Pyrimidines / Q000378:metabolism*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013329:Structure-Activity Relationship; D001618:beta-Lactamases / Q000378:metabolism
D004274:DNA, Recombinant; D004278:DNA, Superhelical; D011687:Purines; D011743:Pyrimidines; D015719:Single-Strand Specific DNA and RNA Endonucleases; D001618:beta-Lactamases