FIG. 7.
Plasmacytoma-cell-specific looping interactions between Ed and the other two enhancers and the promoter. A different restriction enzyme, NspI, was used in this experiment. Quantification of PCR signals with primer 17 as an anchor. The vertical lines indicate the NspI cutting sites that were selected for analysis of the looping interactions. Nineteen fragments were obtained in the region from the promoter to Ed after digestion with NspI. We chose to study the looping interactions within the fragments indicated by the vertical lines corresponding to the promoter of the Vκ19 gene, the three enhancers, the adjacent fragments (so that we could normalize the ligation efficiency between different experiments), and one or two fragments between the enhancers. Sequences complementary to primer 8 are far upstream of MEi (>300 kb) on the κf and κ° alleles of MPC-11 and P815 cells, and sequences complementary to primer 9 are similarly far upstream of MEi (>300 kb) on the κf allele of MPC-11 cells (Fig. 2) (8). Here, the anchor primer and the adjacent primer are pointing towards the other primers instead of having all the primers pointing in the same direction. This configuration, however, does not influence cross-linking frequency estimates because restriction digestion goes to near completion, and those trace products from uncut DNA are much longer than the 3C product and are not seen on gels whatsoever after PCR amplification. We designed our 3C primer pairs to yield products generally between 100 and 250 bp in length, whereas the sizes of the PCR products resulting from primer 17 paired with primers 15 and 14 in uncut DNA would be 0.7 and 3 kb, respectively. These and longer bands are not observed in our experiments. In addition, the primers are in vast molar excess and are not depleted upon the completion of the PCR cycles. Thus, cross-linking frequencies are not being underestimated because of primer competition for PCR products.