Structure of the transcriptionally repressed phosphate-repressible acid phosphatase gene (PHO5) of Saccharomyces cerevisiae.

We developed a high-copy-number plasmid system containing the entire structural and regulatory sequences of the phosphate-repressible acid phosphatase (PHO5) gene and the TRP1/ARS1 replicator sequences of the yeast Saccharomyces cerevisiae to investigate the mechanism of repression-derepression of transcription. The resulting plasmid was used to transform either wild-type cells or a number of strains which contain mutations in various trans-acting regulatory loci for the production of acid phosphatase. Results of analysis of mRNA levels isolated from the transformed strains grown under repressed or derepressed conditions suggested that normal transcriptional regulation of the gene persisted, although gene copy number was significantly increased. Analysis of changes in linking number (i.e., the number of negative supercoils) of the plasmid isolated under repressed and derepressed growth conditions revealed that the transcriptionally inactive plasmid contained approximately three more negative supercoils than the transcriptionally active plasmid. This difference in topological state was similarly seen in a plasmid containing a sequence-related acid phosphatase gene (PHO11) under the same regulatory control system, but it was not seen in plasmids isolated from some strains containing mutations which caused either fully constitutive or nonderepressible production of acid phosphatase. Finally, analysis of the nucleosome positioning along the inactive gene sequence revealed that an abnormally broad internucleosomal spacer is present in a region presumed to function in the regulation of transcription by the level of Pi in the growth media.

Suppression of oncogene-induced transformation by a deletion mutant of c-jun.

Jun and Fos proteins are DNA-binding proteins that are involved in the control of gene expression through transcriptional regulation. We have made a deletion mutant of the c-jun gene that lacks amino acids 3-122 of c-jun, and thus is missing the major transactivation domain of c-jun, but retains the DNA-binding and leucine zipper domains. Unlike c-Jun, the mutant protein is unable to stimulate the transcription of an AP-1 responsive gene, and unlike c-jun this mutant gene is unable to transform rat embryo cells in cooperation with an activated ras gene. However, this mutant protein blocks in vitro DNA binding of Jun-Jun homodimers and Jun-Fos heterodimers, transcriptional activation induced by c-jun or c-fos and transformation of rat embryo cells induced by an activated ras gene and a deregulated c-jun or c-fos gene. In addition, transformation of rat embryo cells induced by an activated ras gene in the presence of the tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) or by ras plus SV40 large T antigen is also inhibited by this dominant-negative mutant, suggesting that a member of the jun or fos family is involved in the pathways leading to transformation in these systems as well. The possible molecular mechanisms by which this dominant-negative mutant of c-jun blocks the functions of wild-type jun and fos family members are discussed.

Early events in the neoplastic transformation of respiratory epithelium.

The complex process of epithelial carcinogenesis is composed of discrete biologic events including the early activation events of "initiation" and "promotion." For lung cancer, these events are only now being elucidated. Despite the identification of possible target genes and their mutations, the "initiation" events for lung cancer remain poorly understood. The identification of these "initiation" events is a crucial step toward the development of practical molecular markers for early detection of this disease. The reversible process of tumor promotion remains somewhat enigmatic but is a promising target for chemoprevention. A wide range of substances, including asbestos and various substances in cigarette smoke, behave as tumor promoters for lung cancer. They appear to promote tumor formation by inducing cellular proliferation mediated in part by growth factors. The intracellular signals these factors provide are ultimately translated into cellular growth via steps involving nuclear transcription factors. Early response genes such as the jun and fos gene family members encode such nuclear transcription factors which are expressed in lung cancer cells and primary bronchial epithelial cells. The expression of these transcription factors is highly responsive to stimulation by growth factors including serum, transforming growth factor, and gastrin-releasing peptide. A more thorough understanding of this process will allow the development of molecular and/or pharmacologic antagonists that can interfere with the biologic process of tumor promotion and therefore function as chemoprevention agents.

Expression of Escherichia coli trp genes and the mouse dihydrofolate reductase gene cloned in Bacillus subtilis.

The mouse dihydrofolate reductase gene and a segment of the Escherichia coli trp operon are expressed in Bacillus subtilis when cloned in the "expression plasmid" pPL608. The cloned mouse gene confers trimethoprim resistance on B. subtilis and the cloned trp fragment complements mutations in the B subtilis trpD, C and F genes Expression of both cloned fragments is dependent on a promoter present in the vector plasmid. The E. coli trp fragment is cloned in a HindIII site within a chloramphenicol acetyltransferase gene present on pPL608, and as a result, expression of the E. coli trpC gene product is inducible by chloramphenicol. The mouse gene is inserted at a PstI site preceding the chloramphenicol acetyltransferase gene and its expression is not chloramphenicol inducible. The replication functions and neomycin-resistance of pPL608 are derived from pUB110. Accordingly, pPL608 is stably maintained at high copy number in B. subtilis.

Constitutive variants of the pC194 cat gene exhibit DNA alterations in the vicinity of the ribosome binding site sequence.

The chloramphenicol-inducible regulation of the expression of cat genes from two Gram-positive bacteria, Staphylococcus aureus and Bacillus pumilus has been suggested to result from the presence of inverted repeat sequences that span the ribosome-binding site (RBS) for cat. In support of this hypothesis, we demonstrate that two derivatives of the pC194 cat gene which are constitutively expressed in Bacillus subtilis are deleted for all or a major portion of the inverted-repeat sequences.

Constitutive cJun expression induces partial macrophage differentiation in U-937 cells.

Previous studies have shown that phorbol ester induced macrophage differentiation in the leukemic cell line U-937 is tightly linked to the expression of the c-jun protooncogene and the generation of AP-1 transcriptional activity. We expressed the c-jun protooncogene in U-937 cells to examine the role of c-jun in the differentiation of these cells. AP-1 DNA binding activity and the expression of AP-1 controlled downstream genes were increased in all clones expressing exogenous cJun. More importantly, these transfectants showed evidence of differentiation as measured by the acquisition of phagocytic capacity, although they did not develop morphological changes associated with differentiation such as adherence. Furthermore, they became committed to terminal differentiation after significantly shorter exposures to phorbol esters than did control cell lines. These data show that cJun expression induces partial differentiation along the macrophage pathway in U-937 cells.

Molecular analysis of the DNA sequences involved in the transcriptional regulation of the phosphate-repressible acid phosphatase gene (PHO5) of Saccharomyces cerevisiae.

The expression of the PHO5 gene of Saccharomyces cerevisiae is transcriptionally regulated in response to the level of inorganic phosphate present in the growth medium. We have identified, by DNA deletion analysis, the sequences (upstream activator sequences) that mediate this response. The sequence 5' CTGCACAAATG 3' is present in two copies located within a 60-base-pair region. The presence of a single copy of the sequence is sufficient for the phosphate-mediated transcriptional response. In addition, a DNA fragment that contains two copies of this sequence will act to repress transcription of a CYC1-lacZ fusion when placed either upstream or downstream of the CYC1 activator sequence.

The role of jun and fos gene family members in 12-O-tetradecanoylphorbol-13-acetate induced hemopoietic differentiation.

Terminal differentiation of the leukemic cell lines U-937 and HL-60 by 12-O-tetradecanoylphorbol-13-acetate is accompanied by marked changes in gene expression. In this study, we demonstrate that the expression of jun and fos gene family members is induced with variable kinetics during 12-O-tetradecanoylphorbol-13-acetate induced differentiation, with c-jun expression best paralleling differentiation. The generation of AP-1 complexes, as measured by DNA binding activity, closely parallels morphological differentiation. Furthermore, the ability of these complexes to regulate gene expression is demonstrated by increased transcription from an AP-1 driven reporter construct and marked increases in the expression of endogenous AP-1 regulated genes. Differentiation assays using water soluble phorbol esters reveal that differentiation becomes irreversible soon after AP-1 appears. This tight correlation between c-jun expression, the generation of AP-1 activity, and differentiation suggests a critical role for this gene and transcriptional complex during this process.