The rfb gene cluster of Salmonella LT2 has been cloned and sequenced. The genes rfbA, rfbB, rfbD, rfbF, rfbG, rfbK, rfbM and rfbP were located individually and the gene rfbL was located outside the cluster. Approximately 16 open reading frames were found in the region which is essential for the expression of O antigen. The gene products of rfbB and rfbG were found to have homology with the group of dehydrogenase and related enzymes described previously. Analysis of the G + C ratio of the rfb cluster extended the area of low-G + C composition previously found in the sequence of rfbJ to the whole rfb gene cluster. Three to five segments with discrete G + C contents and codon adaptation indices are present in the rfb region, indicating a heterogeneous origin of these segments. Potential promoters were found near the start of the rfb region, supporting the possibility that the rfb gene cluster is an operon.

Early growth response factor-1 (Egr-1) binds to the promoters of many genes whose products influence cell movement and replication in the artery wall. Here we targeted Egr-1 using a new class of DNA-based enzyme that specifically cleaved Egr-1 mRNA, blocked induction of Egr-1 protein, and inhibited cell proliferation and wound repair in culture. The DNA enzyme also inhibited Egr-1 induction and neointima formation after balloon injury to the rat carotid artery wall. These findings demonstrate the utility of DNA enzymes as biological tools to delineate the specific functions of a given gene, and implicate catalytic nucleic acid molecules composed entirely of DNA as potential therapeutic agents.

Cleavage of heparan sulfate by the ␤-D-endoglucuronidase heparanase (HPSE) is a fundamental event in a number of important physiological processes including inflammation, wound healing, and angiogenesis. HPSE activity has also been directly correlated with pathological conditions such as tumor growth and metastasis and autoimmune disease. The tight regulation of HPSE expression and function is critical to ensure homeostasis of the normal physiological processes to which it contributes and to prevent imbalance toward pathological situations. Little is known about the transcriptional mechanisms that regulate HPSE expression. In this study we have shown human HPSE gene transcription in Jurkat T cells is induced upon activation. Functional analysis of the HPSE promoter has identified a 280-bp region that is highly inducible. Mutation studies together with supershift experiments have identified a 4-bp motif that binds the transcription factor early growth response-1 (Egr1) and is critical in regulating inducible HPSE gene transcription. Furthermore, the overexpression of Egr1 resulted in the enhanced activation of the HPSE promoter. By using MAPK pathway inhibitors, we have also shown that inducible expression of HPSE mRNA and the activity of the 280-bp HPSE promoter element are dependent on the ERK1/2 (MEK1/2) pathway. This pathway is critical for induction of Egr1 expression at both the mRNA and protein level in T cells, an observation that provides further support to Egr1 playing an important role as a key activator of HPSE expression. In addition, HPSE and Egr1 were shown to co-localize by immunohistochemistry to invading mononuclear leukocytes in actively induced experimental autoimmune encephalomyelitis in rats. These findings provide the first insight into the mechanisms controlling inducible transcription of the HPSE gene, and could represent an important lead into understanding how HPSE expression is deregulated in metastatic tumor cells.Heparan sulfate proteoglycans are key structural components of the extracellular matrix (ECM) 1 and cell surfaces. They comprise a protein core covalently linked to linear chains of the complex sulfated glycosaminoglycan, heparan sulfate (HS) (1-3). The HS side chains of heparan sulfate proteoglycans mediate the assembly and stability of the ECM through interactions with the various matrix components (e.g. collagen, laminin, and fibronectin) (1, 3) and also specifically bind a range of cytokines and growth factors (e.g. basic fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor, and transforming growth factor-␤) thereby functioning as a storage depot for these factors (4, 5). The ␤-D-endoglycosidase heparanase (HPSE) cleaves HS and facilitates the degradation of the ECM and the release of HS-bound growth factors (6, 7). Heparanase is a normal constituent of activated leukocytes, endothelial cells, vascular smooth muscle cells, and cytotrophoblasts; however, its expression is also "hijacked" by metastatic tumor cells (6, 7). The level of HPSE activit...

Cell migration and proliferation that follows injury to the artery wall is preceded by signaling and transcriptional events that converge at the promoters of multiple genes whose products can influence formation of the neointima. Transcription factors, such as early growth response factor-1 (Egr-1), with nucleotide recognition elements in the promoters of many pathophysiologically relevant genes, are expressed at the endothelial wound edge within minutes of injury. The mechanisms underlying the inducible expression of Egr-1 in this setting are not clear. Understanding this process would provide important mechanistic insights into the earliest events in the response to injury. In this report, we demonstrate that fibroblast growth factor-2 (FGF-2) is released by injury and that antibodies to FGF-2 almost completely abrogate the activation and nuclear accumulation of Egr-1. FGF-2-inducible egr-1-promoter-dependent expression is blocked by PD98059, a specific inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK)-1/2 (MEK-1/2), as well as by dominant negative mutants of ERK-1/2. Inducible ERK phosphorylation after injury is dependent on release and stimulation by endogenous FGF-2. Antisense oligonucleotides directed at egr-1 mRNA suggest that Egr-1 plays a necessary role in endothelial repair after denudation of the monolayer. These findings demonstrate that inducible Egr-1 expression after injury is contingent on the release and paracrine action of FGF-2.

Catalytic DNA molecules that target the transcription factor c- jun inhibit skin cancer growth in mice.