The immunoglobulin kappa gene is specifically demethylated during B-cell maturation in a process which utilizes discrete cis-acting modules such as the intronic kappa enhancer element and the matrix attachment region (MAR). While any MAR sequence is sufficient for this reaction, mutation analysis indicates that tissue specificity is mediated by kappaB binding sequences within the kappa intronic enhancer. The plasmacytoma cell line S107 lacks kappaB binding activity and fails to demethylate the kappa locus. However, B-cell specific demethylation is restored by the introduction of an active kappaB binding protein gene relB. This represents the first demonstration of a trans-acting factor involved in cell-type-specific demethylation, and suggests that the same protein-DNA recognition system used for transcription may also contribute to the earlier developmental events that bring about activation of the kappa locus.

Allelic exclusion in light-chain synthesis is thought to result from a feedback mechanism by which the expression of a functional light chain on the surface of the B cell leads to an intracellular signal that down-regulates the V(D)J recombinase, thus precluding rearrangement of the other allele. Whereas such a feedback mechanism clearly plays a role in the maintenance of allelic exclusion, here we provide evidence suggesting that the initial establishment of allelic exclusion involves differential availability of the two alleles for rearrangement. Analysis of + B-cell populations and of individual + B cells that have rearranged only one allele demonstrates that in these cells, critical sites on the rearranged allele are unmethylated, whereas the nonrearranged allele remains methylated. This pattern is apparently generated by demethylation that is initiated at the small pre-B cell stage, on a single allele, in a process that occurs prior to rearrangement and requires the presence in cis of both the intronic and 3 enhancers. Taken together with data demonstrating that undermethylation is required for rearrangement, these results indicate that demethylation may actually underly the process of allelic exclusion by directing the initial choice of a single allele for rearrangement.

We have analyzed the CTF4 (CHL15) gene, earlier identified in two screens for yeast mutants with increased rates of mitotic loss of chromosome III and artificial circular and linear chromosomes. Analysis of the segregation properties of circular minichromosomes and chromosome fragments indicated that sister chromatid loss (1:0 segregation) is the predominant mode of chromosome destabilization in ctf4 mutants, though nondisjunction events (2:0 segregation) also occur at an increased rate. Both inter-and intrachromosomal mitotic recombination levels are elevated in cf4 mutants, whereas spontaneous mutation to canavanine resistance was not elevated. A genomic clone of CTF4 was isolated and used to map its physical and genetic positions on chromosome XVI. Nucleotide sequence analysis of CTF4 revealed a 2.8-kb open reading frame with a 105-kDa predicted protein sequence. The CTF4 DNA sequence is identical to that of POBI, characterized as a gene encoding a protein that associates in vitro with DNA polymerase ao At the N-terminal region of the protein sequence, zinc finger motifs which define potential DNA-binding domains were found. The C-terminal region of the predicted protein displayed similarity to sequences of regulatory proteins known as the helix-loop-helix proteins. Data on the effects of a frameshift mutation suggest that the helix-loop-helix domain is essential for CTF4 function. Analysis of sequences upstream of the CTF4 open reading frame revealed the presence of a hexamer element, ACGCGT, a sequence associated with many DNA metabolism genes in budding yeasts. Disruption of the coding sequence of CTF4 did not result in inviability, indicating that the CTF4 gene is nonessential for mitotic cell division. However, c1f4 mutants exhibit an accumulation of large budded cells with the nucleus in the neck. ctf4 rad52 double mutants grew very slowly and produced extremely high levels (50%) of inviable cell division products compared with either single mutant alone, which is consistent with a role for CTF4 in DNA metabolism.

Studies on the mechanisms of inducible and constitutive activity of NF-B transcription factors have been hampered by the lack of appropriate mutant cell lines. We have analyzed the defect in the murine S107 plasmacytoma cell line, which was previously found to lack both constitutive and inducible NF-B activity. Our analysis shows that these cells bear a specific defect that interferes with NF-B induction by many diverse stimuli, such as lipopolysaccharide, phorbol 12-myristate 13-acetate, UV light, x-rays, and H 2 O 2 . This does not however represent a general signal transduction defect, because AP-1 transcription factors are readily induced by the same stimuli. Phosphatase inhibitors such as okadaic acid as well as calyculin A can efficiently induce NF-B in S107 cells via a pathway apparently insensitive to the radical scavenger pyrrolidine dithiocarbamate. Furthermore, MEKK1 a protein kinase supposedly induced by some of the above stimuli, is also capable of activating NF-B. Interestingly, both the potent physiological inducer of NF-B TNF␣ as well as endoplasmic reticulum overload can induce NF-B via a PDTC sensitive pathway. In all cases, DNA-binding NF-B complexes are comprised predominantly of p50-RelA heterodimers, and NF-B activation results in the induction of transiently transfected or resident reporter genes. In summary, these results suggest that the pathways for many NF-B-inducing stimuli converge at a specific junction, and this pivotal step is mutated in the S107 cell line. Yet there are alternative routes bypassing this critical step that also lead to NF-B induction. These routes utilized by tumor necrosis factor ␣ and endoplasmic reticulum overload are still intact in this cell line.The NF-B/Rel transcription factors consist of five mammalian family members that bind to DNA as homo-and/or heterodimers (1-4). Two of the family members, NFKB1 and NFKB2, are synthesized as precursor proteins (p105 and p100, respectively), which are proteolytically processed to obtain the mature proteins p50 and p52. The other three members, RelA, RelB, and c-Rel, are produced directly without such a processing step. These latter family members contain efficient transactivation domains in their COOH-terminal domains (RelA and c-Rel) or both NH 2 -and COOH-terminal domains (RelB), and therefore they are largely responsible for the transactivation capacity of homo-and heterodimers containing these subunits.NF-B was originally identified as a transcription factor constitutively present in the nuclei of mature B and plasma cells, yet present in a latent but inducible form in pre-B cells as well as most other cell types (5-7). The mechanism leading to this inducibility was resolved to some extent. In most cell types, NF-B family members are localized in the cytoplasm due to the interaction with inhibitory proteins, the I Bs (8). These I B proteins first of all inhibit nuclear translocation of NF-B proteins by masking the nuclear localization signal and also directly block their DNA binding. The I B proteins again represe...

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