Two forms of FosB transcript and their products can be identified in mouse NIH 3T3 cells following serum induction. The larger RNA codes for a 338-amino acid protein, whereas the smaller RNA results from the removal of an additional 140 nucleotides from FosB mRNA by alternative splicing. This alternative splicing event places a stop codon following the "leucine zipper" region and results in a shorter protein (FosB2) of 237 amino acids that lacks 101 amino acids at the carboxyl terminus. FosB2 is able to form heterodimers with c-Jun and bind to an AP-1 site but is not able to activate the transcription of promoters containing AP-1 sites. Furthermore, FosB2 can not only suppress the transcriptional activation by c-Fos and c-Jun of promoters containing an AP-1 site but also interferes with the transforming potential of viral and cellular Fos proteins. We propose that FosB2 protein functions as a trans-negative regulator.

Using proliferating cell nuclear antigen affinity chroma-tography and glycerol gradient centrifugation of partially purified fractions from mouse FM3A cells we have been able to isolate novel complexes of DNA polymerase delta and DNA ligase 1 containing clearly defined subunit compositions. In addition to the well known catalytic subunit of 125 kDa and accessory subunit of 48 kDa, the DNA polymerase delta complex contained three supplementary components, one of which reacted with antibodies directed against the p40 and p37 subunits of RF-C. Of the two remaining components, one termed p66 turned out to be coded by a gene whose putative C-terminal domain displayed significant homology with that of the Cdc27 subunit of Schizosaccharomyces pombe polymerase delta. On the basis of these and other observations, we propose p66 to be the missing third subunit of mammalian DNA polymerase delta. The DNA ligase 1 complex was made up of three novel components in addition to the 125 kDa catalytic subunit, two of which, p48 and p66, were common to DNA polymerase delta. We discuss the implications of our findings within the current framework of our understanding of DNA replication.

We have studied the phosphorylation of the nuclear oncoprotein Fos by cyclic AMP-dependent protein kinase (PKA). We demonstrate that the human c-Fos protein, phosphorylated either in vitro with purified PKA 65 kDa (3,12). This heterogeneity is largely due to phosphorylation of serine residues (3). Although both c-Fos and its viral homolog v-Fos are phosphorylated, the cellular protein is more extensively phosphorylated (3). Major phosphorylation sites in c-Fos have been localized to its C terminus within the amino acid sequence that differs from that of v-Fos (3, 41). Precise identification of the serine residues phosphorylated, as well as the kinases involved in this phosphorylation, would help our understanding of the biological function of Fos phosphorylation.A large body of data implicates the involvement of the cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway in the regulation of both the fos gene and the Fos protein (31). Activation of the PKA pathway by adenyl cyclase agonists such as forskolin leads to Fos expression (2,7,8,21,26), presumably via interaction of the phosphorylated cAMP responsive element (CRE)-binding protein with the CRE in the fos promoter (25,36 PKA does phosphorylate the human c-Fos protein in vitro on a site also phosphorylated in vivo. We have identified serine residue 362 as the phosphorylated amino acid. Mutation of serine residues 362 to 364 to alanine enhances the transforming activity of c-Fos to levels comparable with those of v-Fos. It has previously been shown that lack of phosphorylation of c-Fos at its C terminus abolishes its ability to downregulate its own promoter but has no effect on transactivation of genes linked to an AP-1 site (29, 36). Our results suggest that part of the mechanism that leads to cell transformation by Fos might be attributed to the loss of a PKA phosphorylation site in v-Fos. Moreover, our results implicate PKA in regulation of Fos activity in a complex manner by controlling both induction and repression of the fos promoter. MATERIALS AND METHODSCell lines and culture conditions. The human choriocarcinoma cell line JEG3 (20)

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We examined DNA methylation and DNase I hypersensitivity of the a-fetoprotein (AFP) and albumin gene region in hepatoma cell lines which showed drastic differences in the level of expression of these genes. We assayed for methylation of the CCGG sequences by using the restriction enzyme isoschizomers HpaH Many studies with different experimental systems have been carried out to elucidate the role that methylation of cytosine bases and change in chromatin structure might play in the regulation of gene expression (for reviews, see references 34 and 35).Controversial results concerning the relationship between the DNA methylation pattern and gene activity have been reported showing either a good correlation (23,47) or no correlation at all (13,46,57) between undermethylation and expression of a specific gene. Various studies involving in vitro methylation of a specific regulatory region of a gene followed by its introduction into eucaryotic cells revealed that DNA methylation does affect transcription (6,17,41,42). Recent studies indicate that undermethylation of only certain critical sites located in the 5' region of genes coding for tissue-specific proteins is necessary but not sufficient for their expression (24,30,56 (48,50). Of these variants, we chose to study an

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By use of different restriction enzymes sensitive to internal cytosine methylation (HpaII, AvaI, HhaI) we have analysed the methylation patterns of albumin and AFP genes in tissues and cell lines with high (liver, yolk sac, hepatoma cell lines), low (fetal and neonatal kidney) or undetectable (spleen, JF1 fibroblasts) expression of either gene. We show that expression of the AFP gene is associated to the demethylation of a whole region or domain extending from -4 to +3 Kb. Moreover, demethylation of a site located at the upstream limit of this domain appears to be correlated with the commitment of the cell type to synthesize AFP. As concerns the albumin gene, we show that the domain in which demethylation is correlated with active gene transcription in hepatoma cell lines has different borders than in tissue. This difference might be related to the different amounts of mRNA synthesized or to an alteration in gene regulation in tumor cells. Finally, we show that low expression of albumin and AFP genes in fetal and neonatal kidney is not correlated with domain demethylation, suggesting that the regulatory mechanisms of expression of these genes are different in kidney as compared with liver.