Decreased immunoglobulin deposition in tumors and increased immature B cells in p53-null mice.

Recent studies have hinted that there may be a relationship between p53 and the immune response. In preliminary experiments, we found significantly decreased levels of immunoglobulin deposition in 13 of 16 p53-null tumors compared with 2 of 17 tumors derived from p53 +/- mice. We further explored the effect of p53 on B-cell development and function. p53-null mice contained more splenic white pulp and more immature B cells in the bone marrow compared with p53 +/- mice. p53-null B cells were hyperresponsive to proliferative challenge but were not more resistant to signal-induced apoptosis. Several p53 DNA-binding sites were localized to the regulatory regions of immunoglobulin heavy and light chain genes, including the KII site, which serves as an enhancer for rearrangement of the mouse kappa chain J cluster genes. Levels of p53 protein and the kappa chain sterile transcript increased after exposure of pre-B cells to the DNA damaging agents etoposide and Adriamycin. Our observations suggest that p53 may be involved in B-cell maturation and may relay certain stress signals to affect B-cell function.

Studies of in vitro transcription by calf thymus RNA polymerase II using a novel duplex DNA template.

Addition of 10 to 100 oligodeoxycytidylate residues to the 3'-OH termini of T7 bacteriophage DNA produces a highly efficient template for transcription in vitro with purified calf thymus RNA polymerase II. Transcription initiates rapidly and selectively at the oligo (dC) ends of such a template and essentially all of the active RNA polymerase II molecules are then committed to a long period of RNA chain elongation. This allows the direct study of the RNA chain elongation and termination reactions and also permits determination of the concentration of active RNA polymerase II that is present. From 15 to 25% of the RNA polymerase molecules in our current preparations are active in these reactions. RNA chain elongation by calf thymus RNA polymerase II is relatively slow (7 nucleotides/s) even at saturating substrate concentrations. The in vitro elongation process appears to be discontinuous, with elongating polymerase molecules pausing for significant periods at certain sequences along the DNA. There is a low, but measurable frequency of RNA chain termination at some sites; however, the majority of elongating transcripts can grow to large sizes (over 6000 nucleotides). Surprisingly, over 60% of the active calf thymus RNA polymerase II molecules form a long DNA-RNA hybrid during in vitro transcription and displace the nontranscribed DNA from the template to produce a characteristic split end structure. DNA-RNA hybrids are also formed during transcription by RNA polymerase II from duplex DNA templates lacking 3' oligo(dC) tails, which takes place predominantly at single strand breaks or ends. Thus the transcriptional elongation reaction carried out by calf thymus RNA polymerase II in vitro differs in several respects from that which must take place in vivo.