A series of Rk-Luc plasmids was constructed carrying the human Rk gene 5′ flanking sequences inserted upstream of the firefly luciferase gene in the pLuc vectors.
21 To make the ends of the human genomic sequence compatible with the polylinker region of the pLuc vectors, the 5′ ends of the 2.0-kb human
EcoRI-
SmaI and 0.2-kb
ApaI-
SmaI fragments were modified by insertion of a unique
XhoI recognition sequence into either the
EcoRI or
ApaI site in p-2.0. The 2.0- or 0.2-kb
XhoI-
SmaI cassettes were then retrieved and inserted in forward or reverse orientation upstream of the luciferase gene in pXP1 or pXP2 to generate p-2.0Luc, p-2.0Luc(−), p-0.11Luc, and p-0.11Luc(−). Plasmids p-2.0h1Luc and p-0.11h1Luc were constructed from p-2.0Luc and p-0.11Luc, respectively, by replacing the wild-type H1 motif (TC
TAATC; −29 to −23) with an inactive h1 sequence (AG
ATCTC).
25 26 Mutagenic oligonucleotide pairs corresponding to positions −36 through −4 were used in conjunction with a
pfu-mediated mutagenesis kit (QuickChange; Stratagene) to introduce the substitutions into this region. After sequencing the segment to ensure accurate mutagenesis, the mutagenized region was then used to replace the corresponding wild-type segment to generate the final mutant Rk-Luc plasmids. An additional series of truncated Rk-luciferase constructs without segments of the 5′ end of the human flanking sequence was generated by unidirectional partial exonuclease III/S1 digestion starting from p-2.0Luc. After linearization with two adjacent restriction enzymes,
XhoI and
BglII, and fill-in protection of the
BglII site with α-phosphorothioate dNTPs, the DNA was nuclease treated (Erase-a-Base kit; Promega) for various periods and recircularized for cloning. Nested deletions of the 2.0-kb region with segments missing from the 3′ end were generated by nuclease degradation, starting with
SalI-
HindIII–digested p-2.0Luc protected at the
HindIII end.