The P4 promoter of the parvovirus minute virus of mice contains a single degenerate GC box sequence which binds the transcription factor Spl with high affinity. The two protomers of murine Spl were affinity purified, and their interactions with the P4 promoter were examined. Several unusual features were observed. Methylation interference experiments demonstrated that Spl makes contacts with both DNA strands, including the central guanine as well as an adenine residue on the cytidine-rich

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... of the GC box. UV photocrosslinking revealed that the 95and the 105-kDa protomers cross-link exclusively to opposite strands of the GC box. These results suggest that the phosphorylation of the 95-kDa Spl protomer results in a change in the way Spl is positioned on the P4 GC box and identifies a high-affinity GC box motif. * Corresponding author. MATERIALS AND METHODS Plasmids and oligonucleotides. The plasmid pMB415 was made by ligating the BamHI-PstI P4 promoter-containing fragment from MVM(p) into the Bluescript KS(+) vector. The plasmid pP4was constructed by ligating the BamHI-PstI P4 promoter-containing fragment into the BS KS (-) vector. The SV40 early promoter was derived from the plasmid pSV2XCAT which has an XbaI linker placed in the AccI site in pSV2CAT. Chromatography resins. WGA sepharose was prepared by coupling wheat germ agglutinin (WGA) (Sigma) to commercially available CNBr-activated Sepharose CL-4B (PL Biochemicals) according to the manufacturer's instructions. The final amount of WGA coupled was 5 mg/ml of resin. Oligonucleotide Sepharose was prepared by coupling the oligonucleotide duplex BG 1/2 (containing the sequence from nucleotide [nt] 137 to nt 173 with three non-base-paired adenines at its left end [ Fig. 1A ]) to CNBr-activated Sepharose CL-4B according to the protocol of Amdt-Jovin et al. (3) . The final concentration of oligonucleotide coupled was 100 ,ug/ml of resin. Cell culture and nuclear extracts. Murine A92L fibroblasts were grown in spinner flasks in Jokliks Dulbecco modified Eagle medium (GIBCO-BRL) supplemented with 5% fetal calf serum. The cells were harvested at a density of 5 x 105, and nuclear extracts were prepared according to the method of Dignam (1) except for the following modifications. All buffers contained the following protease inhibitors: phenylmethylsulfonyl fluoride, metabisulfite, and benzamidine at 0.5 mM; EDTA and EGTA [ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid] at 0.1 mM; and aprotinin, leupeptin, and pepstatin at 50 ,ug/ml]. The cells were lysed in hypotonic buffer yethylpiperazine-N'-2-ethanesulfonic acid; pH 7.9], 10 mM KCl, 1.5 mM MgCl2, 20% glycerol) that also contained 1% (vol/vol) Nonidet P-40 (NP-40; Calbiochem) and 2% (wt/vol) polyvinyl alcohol (PVA) (Fluka) to stabilize the nuclei. The nuclei were pelleted at 10,000 x g for 15 min and gently resuspended in high-salt buffer (0.6 M KCl, 20 mM HEPES, 1.5 mM MgCl2, 20% glycerol, 0.1% NP-40, 10% [wt/vol] sucrose [Schwartz-Mann]). The nuclei were extracted on a nutator for 1 h at 4°C. The nuclear debris was removed by centrifuging at 80,000 x g in a SW-50 rotor for 30 min. The supernatant was dialyzed against 100 volumes of buffer D (20 6661 on May 10, 2020 by guest