The ability of Listeria monocytogenes to move within the cytosol of infected cells and their ability to infect adjacent cells is important in the development of infection foci leading to systemic disease. Interaction with the host cell microfilament system, particularly actin, appears to be the basis for propelling the bacteria through the host cell cytoplasm to generate the membraneous protrusions whereby cell‐to‐cell spread occurs. The actA locus of L.monocytogenes encodes a 90 kDa polypeptide that is a key component of bacterium‐host cell microfilament interactions. Cloning of the actA gene allowed the identification of its gene product and permitted construction of an isogenic mutant strain defective in the production of the ActA polypeptide. Sequencing of the region encoding the actA gene revealed that it was located region encoding the actA gene revealed that it was located between the metalloprotease (mpl) and phosphatidylcholine‐specific phospholipase C (plcB) genes. Within the cytoplasm of the infected cells, the mutant strain grew as microcolonies, was unable to accumulate actin following escape from the phagocytic compartment and was incapable of infecting adjacent cells. It was also dramatically less virulent, demonstrating that the capacity to move intracellularly and spread intercellularly is a key determinant of L.monocytogenes virulence. Like all other virulence factors described for this microorganism, expression of the ActA polypeptide is controlled by the PrfA regulator protein. The primary sequence of this protein appeared to be unique with no extended homology to known protein sequences. However, an internal repeat sequence showed strong regional homology to a sequence from within the hinge region of the cytoskeletal protein vinculin.

The prfA gene ofListeria monocytogenes encodes a protein that activates transcription of the listeriolysin gene (lisA). In order to explore the role of the prfA gene product in the pathogenesis of listerial infection, we constructed a site-directed insertion mutation in prfA by the chromosomal integration of a novel suicide vector containing a portion of the prfA coding region. This mutation not only transcriptionally silenced the listeriolysin (lisA) gene but also abrogated production of specific RNA transcripts corresponding to the phosphatOdylfiititol-specific phospholipase C (pic) and metalloprotease (mpl) genes, two further virulence gene products expressed only by pathogenic Listeria strains. The strain was also found to be avirulent when tested in a mouse model of listerial infection. The concomitant loss of multiple characteristics such as production of LisA, Pic, Mpl, and loss of virulence in a mouse infection model is the result of a mutation in a single gene and demonstrates that the prfA gene product is a positive regulator of multiple virulence determinants in L.monocytogenes.

cancer ͉ gene expression ͉ myc oncogene ͉ transcriptional control ͉ tumor suppressor

The c-myc protooncogene encodes a transcription factor (Myc) with oncogenic potential. Myc and its dimerization partner Max are bHLH-Zip DNA binding proteins controlling fundamental cellular processes. Deregulation of c-myc leads to tumorigenesis and is a hallmark of many human cancers. We have identified and extensively characterized ancestral forms of myc and max genes from the early diploblastic cnidarian Hydra, the most primitive metazoan organism employed so far for the structural, functional, and evolutionary analysis of these genes. Hydra myc is specifically activated in all stem cells and nematoblast nests which represent the rapidly proliferating cell types of the interstitial stem cell system and in proliferating gland cells. In terminally differentiated nerve cells, nematocytes, or epithelial cells, myc expression is not detectable by in situ hybridization. Hydra max exhibits a similar expression pattern in interstitial cell clusters. The ancestral Hydra Myc and Max proteins display the principal design of their vertebrate derivatives, with the highest degree of sequence identities confined to the bHLH-Zip domains. Furthermore, the 314-amino acid Hydra Myc protein contains basic forms of the essential Myc boxes I through III. A recombinant Hydra Myc/Max complex binds to the consensus DNA sequence CACGTG with high affinity. Hybrid proteins composed of segments from the retroviral v-Myc oncoprotein and the Hydra Myc protein display oncogenic potential in cell transformation assays. Our results suggest that the principal functions of the Myc master regulator arose very early in metazoan evolution, allowing their dissection in a simple model organism showing regenerative ability but no senescence.cell proliferation | cnidaria | development | transcription factor

In a fluorescence polarization screen for the MYC-MAX interaction, we have identified a novel small-molecule inhibitor of MYC, KJ-Pyr-9, from a Kröhnke pyridine library. The K d of KJ-Pyr-9 for MYC in vitro is 6.5 ± 1.0 nM, as determined by backscattering interferometry; KJ-Pyr-9 also interferes with MYC-MAX complex formation in the cell, as shown in a protein fragment complementation assay. KJ-Pyr-9 specifically inhibits MYC-induced oncogenic transformation in cell culture; it has no or only weak effects on the oncogenic activity of several unrelated oncoproteins. KJ-Pyr-9 preferentially interferes with the proliferation of MYC-overexpressing human and avian cells and specifically reduces the MYC-driven transcriptional signature. In vivo, KJ-Pyr-9 effectively blocks the growth of a xenotransplant of MYC-amplified human cancer cells.M YC is a transcriptional regulator that occupies an apex position in the organizational hierarchy of the cell (1-3). It belongs to a family of basic helix-loop-helix leucine zipper (bHLH-LZ) proteins that dimerize with the small bHLH-LZ protein MAX to become functional (4). The MYC-MAX heterodimer preferentially binds to the palindromic DNA sequence CACGTG, referred to as the E-box motif. As a transcription factor, MYC can bind to the promoters of target genes to stimulate or repress transcriptional activity (5-7). The human genome contains three MYC genes, c-MYC, N-MYC, and L-MYC. Throughout this paper, we will use "MYC" to indicate the protein product of the c-MYC gene.MYC is involved in almost all cancers (8, 9). It is rarely mutated, but achieves gain of function through overexpression or amplification. Because of this broad pathogenic significance, MYC is an important cancer target. However, both conceptual and practical difficulties have stood in the way of identifying potent and effective small-molecule inhibitors of MYC. The conceptual obstacles reflect concern about inhibiting a gene that controls essential cellular activities. Because MYC plays an important role in cell proliferation (10, 11), it is often argued that inhibition of this function would lead to broad and unacceptable side effects in vivo. However, studies with the dominant-negative MYC construct Omomyc have shown that inhibiting MYC has only mild and rapidly reversible effects on normal, fast-proliferating tissues (8,12,13). The main practical difficulty in targeting MYC is the absence of pockets or grooves that could serve as binding sites for small molecules (14).The preferred strategy for the identification of potential MYC inhibitors has been interference with MYC-MAX dimerization (15-18). The formation of the MYC-MAX heterodimer involves the bHLH-LZ domains of the two partner molecules with a protein-protein interaction (PPI) surface of ∼3,200 Å 2 . This surface lacks well-defined binding sites for small molecules and therefore is widely considered as "undruggable." However, despite the large interaction surface, a single-amino acid substitution can completely disrupt the dimerization of MYC with MAX (14...