Karin Johansson

REAL-TIME ANALYSIS OF OCT PROTEIN-OCTAMER INTERACTION AND TRANSCRIPTION COMPLEX ASSEMBLY

Specific interactions between the protein-binding sequence of the immunoglobulin transcription regulatory element, the octamer, and Oct proteins have been investigated using a biosensor based on surface plasmon resonance. By analysis of in vitro translated Oct1 and Oct2A with a consensus octamer probe, it was shown that the affinity constant, association rate constant and dissociation rate constant of Oct1 were higher than for Oct2A. The biggest difference was in the association rate constants, but this difference was reduced when an octamer motif containing a point mutation was used as a probe. Elements in the octamer flanking sequence could increase the on-rate of Oct proteins to a mutated octamer while not decreasing the off-rate. Oct-octamer interaction in whole nuclear extracts could be detected readily in the biosensor and adapter interactions with template bound proteins were revealed. Thus, biosensor analysis represent a fast and convenient alternative approach to study specific protein-DNA and protein-protein interactions in analysis of transcriptional regulation.

Analysis of Oct2-Isoform Expression in Lipopolysaccharide-Stimulated B Lymphocytes

Oct2‐isoform expression in splenic B cells stimulated with lipopolysaccharide or lipopolysaccharide plus phorbol‐di‐butyrate was analysed by cDNA cloning. The frequency of Oct2‐positivc clones was 1/15, 000 in both libraries. Two new isoforms were found that generate novel amino‐ or carboxy‐terminal sequences. An isoform lacking exon 11 destroyed the carboxy‐terminal leucin‐zipper region and introduced a frame shift creating a novel, proline‐rich carboxy terminus. A new exon containing a highly basic region (4c) was characterized, between exons 4 and 5. This exon was inserted between glutamine‐rich regions 2 and 3, carboxy terminal of a tentative leucine‐zipper structure. In addition, a new combination isoform containing Oct2as amino terminal insert (exon 7a) and Oct2bs carboxy terminal insert (exon 13) was found that created a novel large isoform, Oct2ab. More frequent use of the classical Oct2a and Oct2b isoforms was observed in the lipopolysaccharide‐stimulated B cells, while a preference for the Oct2ab and Oct2ba isoforms was observed in lipopolysaccharide plus phorbol‐di‐butyrate‐lreated cells.