Figure 2.

ORF1p polymers—effect of oligonucleotides and NaCl. Cross-linking with EGS was carried out as described in the ‘Materials and Methods’ section. (A) Cross-linked ORF1p products generated by 0.05 mM and 1.0 mM EGS at 0.05 and 0.5 M NaCl in the absence and presence of a molar excess of oligonucleotides. The 29-mer single-stranded DNA is d29_c; the 53-mer is d53_pT-b (Table 1). The numbers to the left of the arrowheads at lane 1 indicate the estimated number of monomers in each species. The estimated kDa's for bands 1–7 are respectively: 41, 85, 127, 163, 216, 229 and 298. The kDa of the marker bands (mk) are: 200, 116, 97, 66, 55, and 37. Estimated kDa >200 are extrapolated values and therefore only approximations. The arrowheads to the right of lane 13 indicate the position of the trimer and a putative dimer of the trimer (trimer₂). (B) Effect of time of addition of oligonucleotide or 0.5 M NaCl on cross-linked products generated by 1 mM EGS. The reactions with oligonucleotides were in 0.05 M NaCl. The single-stranded DNA is d29; the double-stranded DNA is a duplex of d29 and d29_c (Table 1). The marker bands are 200, 116 and 97 kDa. One millimolar EGS was added immediately after NaCl or oligonucleotides were added to the reactions electrophoresed in lanes 9–14. (C) Effect of oligonucleotide length on ORF1p polymerization. Cross-linking was with 1 mM EGS. The oligonucleotides used in these experiments are all of the dN_c set shown in Table 1 where N is the length (L) of the oligonucleotide. Each oligonucleotide was tested at 1 and 0.5 µM, as indicated in the major header (length/µM) for lanes 3–13 in reactions that contained 0.05 M NaCl. The marker bands are 200, 116 and 97 kDa.