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GAL4 is a classically defined positive regulatory gene controlling the five inducible structural genes of galactose/ melibiose utilization in yeast. The positive regulatory function of the GAL4 gene product in turn is controlled by the product of another gene, the negative regulator GAL80. We have cloned a 3. 1-kilobase fragment containing GAL4 by homologous complementation using the multicopy chimeric vector YEp24 and demonstrated that multiple copies of GAL4 in yeast have pronounced dosage effects on the expression of the structural genes. Yeast transformed with GAL4-bearing plasmid become constitutive for expression of the galactose/melibiose genes, even in normally repressing (glucose) medium. Multiple copies ofthe GAL4 plasmid also increase expression of the structural genes in inducing (galactose) medium and can partially overcome the effects of a dominant super-repressor mutant, GAL80S. Using an internal deletion in GAL4, we have demonstrated that these dosage effects are due to overproduction of GAL4 positive regulatory product rather than an effect of the flanking sequences titrating out a negative regulator. These results point to the importance of competitive interplay between the positive and negative regulatory proteins in the control of this system. We have also used the dosage effect of GAL4 plasmid in combination with different GAL4 and GAL80 alleles to create new phenotypes. We interpret these phenotypes as indicating that (i) the repressing effects of glucose, at least in part, are mediated by the product ofthe negative regulatory gene, GAL80, and (ii) the GAL80 protein may have specific interactions with the control regions of the structural genes.A well-defined system for delineating aspects ofeukaryotic regulation is the galactose/melibiose utilization regulon in the yeast Saccharomyces cerevisiae. The GAL4 gene in this system encodes a positive regulatory protein required to express transcriptionally the structural genes for galactose/melibiose metabolism (1-3): GAL1 (galactokinase, EC 2.7.1.6), GAL7 (a-Dgalactose-l-phosphate uridyltransferase, EC 2.7.7.12), GALIO (uridine diphosphoglucose 4-epimerase, EC 5.1.3.2), and MEL1 (a-galactosidase, EC 2.1.1.22) (4, 5). GAL4 controls expression at transcription (3, 6-8). The system also involves a negative regulatory gene, GAL80, which codes for a protein that prevents expression of the structural genes in the absence of inducer (9). At least three allelic states exist for both GAL4 and GAL80 (Table 1).Douglas and Hawthorne (10) proposed an operator/repressor control circuit for the galactose regulon, analogous to bacterial systems. In their model, GAL80 protein ("i protein" by their nomenclature) represses GAL4 transcription under noninducing conditions by binding at the operator for GAL4. Induction involves release of GAL80 repressor, de novo GAL4 transcription and translation, and GAL4 protein-mediated transcription of the structural genes. However, later experiments have forced a revision ofthis model. First, different allelic combinat...

Breast cancer metastasis suppressor 1 (BRMS1) suppresses metastasis of multiple human and murine cancer cells without inhibiting tumorigenicity. By yeast two-hybrid and co-immunoprecipitation, BRMS1 interacts with retinoblastoma binding protein 1 and at least seven members of the mSin3 histone deacetylase (HDAC) complex in human breast and melanoma cell lines. BRMS1 co-immunoprecipitates enzymatically active HDAC proteins and represses transcription when recruited to a Gal4 promoter in vivo. BRMS1 exists in large mSin3 complex(es) of ϳ1.4 -1.9 MDa, but also forms smaller complexes with HDAC1. Deletion analyses show that the carboxyl-terminal 42 amino acids of BRMS1 are not critical for interaction with much of the mSin3 complex and that BRMS1 appears to have more than one binding point to the complex. These results further show that BRMS1 may participate in transcriptional regulation via interaction with the mSin3⅐HDAC complex and suggest a novel mechanism by which BRMS1 might suppress cancer metastasis.The complex process of cancer cell dissemination and the establishment of secondary foci involves the acquisition of multiple abilities by metastatic cells. For example, blood-borne metastasis requires cells to invade from the primary tumor, enter the circulation, survive transport, arrest at a secondary site, recruit a blood supply, and proliferate at that site (1). The ability to accomplish all of these steps likely involves changes in, and coordinated expression of, a large assortment of genes. Consistent with this notion, several genes, proteins, and pathways have been associated with metastatic progression, including oncogenes, motility factors, and matrix metalloproteinases (1, 2). In addition to metastasis-promoting genes, a new class of molecules called metastasis suppressors has been described (reviewed in Refs. 2-5). By definition, metastasis suppressors inhibit metastasis without blocking primary tumor growth, presumably by inhibiting one or more steps necessary for metastasis. To date, 13 metastasis suppressor genes have been identified that reduce the metastatic ability of cancer cell line(s) in vivo without affecting tumorigenicity, namely breast cancer metastasis suppressor 1 (BRMS1), 1 CRSP3, DRG1, KAI1, KISS1, MKK4, NM23, RhoGDI2, RKIP, SSeCKs, VDUP1, E-cadherin, and TIMPs (reviewed in Refs. 4 and 5).We identified BRMS1 using differential display to compare highly metastatic breast carcinoma cells with related but metastasis-suppressed cells (6). Enforced expression of BRMS1 suppressed metastasis in three animal models, namely human breast (6), murine mammary (7), and human melanoma cells (8). Additionally, BRMS1 mapped to loci in murine (7) and human (6) genomes that had previously been implicated in metastasis control (9). The BRMS1 protein localized to nuclei and restored gap junctional intercellular communication in both breast and melanoma tumor cell lines (8,10,11), but its molecular functions remain to be elucidated.One approach to determine a mechanism of action involves identifying which...

Galactose-inducible genes (GAL genes) in yeast Saccharomyces cerevisiae are efficiently transcribed only when the sequencespecific transcription activator Gal4p is activated. Activation of Gal4p requires the interaction between the Gal4p inhibitory protein Gal80p and the galactokinase paralog, Gal3p. It has been proposed that Gal3p binds to a Gal80p-Gal4p complex in the nucleus to activate Gal4p. Here, we present evidence that the Gal3p-Gal80p interaction occurs in the cytoplasm, and concurrently, Gal80p is removed from Gal4p at the GAL gene promoter. We also show that GAL gene expression can be activated by heterologous protein-protein interaction in the cytoplasm that is independent of galactose and Gal3p function. These results indicate that galactose-triggered Gal3p-Gal80p association in the cytoplasm activates Gal4p in the nucleus.