FIG. 4.

Trimerization and an intact B box are required for the association of NF7 with RNAPII active transcriptional units. (A) Transcripts of several modified forms of HA-tagged NF7 were injected into the cytoplasm of stage V oocytes, and nuclear spreads were prepared 18 h later. Newly made proteins (in red) were detected with anti-HA MAb 3F10 and an Alexa 594-conjugated secondary antibody. In all preparations, DNA was counterstained with picogreen (nucleoli and chromosomal axes are the only structures labeled) and the merged images are presented. A differential interference contrast image and its corresponding fluorescence micrograph are presented for ΔCC284-409 to emphasize the fact that while chromosomal loops are present, they are not labeled by MAb 3F10. Note that the two domains required for chromosomal association are the B box (ΔN275) and the coiled coil (ΔCC284-409). CBs, which were found to accumulate several of the newly expressed proteins, are indicated by arrows. Scale bars are10 μm. (B) Western blot analysis of the newly expressed protein with MAb 3F10. Each lane corresponds to 10 nuclei of stage V oocytes. The values on the left are molecular sizes in kilodaltons.