Regulation of cytomegalovirus gene expression: alpha and beta promoters are trans activated by viral functions in permissive human fibroblasts.

We have fused immediate (alpha) and delayed (beta) early promoter-regulatory sequences taken from the cytomegalovirus (CMV) genome to Escherichia coli lacZ (beta-galactosidase) as an indicator gene to study regulated expression of these promoters. After transfection of human fibroblast cells with plasmid constructs carrying beta-galactosidase fusions, and subsequent infection with CMV, we have demonstrated that viral trans-acting functions up-regulate the expression of these genes in a temporally authentic manner. The alpha promoter is activated even when de novo protein synthesis is blocked and when UV-inactivated virus is used, suggesting that, as for herpes simplex virus type 1 (HSV-1), a virion structural protein is responsible for its up-regulation. We have found that HSV-1, as well as CMV, is capable of trans activating the CMV alpha promoter. The beta promoter is activated by CMV but is completely unresponsive to HSV-1 infection. The temporal synthesis of the alpha and beta promoters in the transient expression system conforms with their natural regulation during viral replication. The beta-galactosidase fusions we describe provide a most exquisitely sensitive indicator system for the study of cis- and trans-acting viral regulatory functions.     

The use of radiation-induced bacterial promoters in anaerobic conditions: a means to control gene expression in clostridium-mediated therapy for cancer

Nuyts, S., Van Mellaert, L., Theys, J., Landuyt, W., Lambin, P. and Anné, J. The Use of Radiation-Induced Bacterial Promoters in Anaerobic Conditions: A Means to Control Gene Expression in Clostridium-Mediated Therapy for Cancer. Radiat. Res. 155, 716-723 (2001). Apathogenic clostridia, which have been genetically engineered to express therapeutic genes, will specifically target hypoxic and necrotic regions in tumors. This specificity can be improved further if the expression of these genes is controlled by a radiation-induced promoter, leading to spatial and temporal control of gene expression. We isolated two radiation-inducible genes of the SOS repair system of Clostridium. Northern blot experiments confirmed radiation activation of the recA and recN genes at a dose of 2 Gy. The promoter region of these genes was isolated and used to regulate expression of the lacZ gene under anaerobic conditions. For the recA promoter, a significant increase of beta-galactosidase activity of 20-30% was seen after 2 Gy irradiation. The recN promoter did not show a significant induction and had a 50-100 times lower basal expression. Treatment of the recombinant clostridial cultures with the cytostatic agent mitomycin C also resulted in a significant increase of beta-galactosidase activity that was under the control of recA or recN promoter. Oxygen does not appear to be necessary in the activation of the SOS repair system by irradiation as tested with Escherichia coli since recA-deficient and recA-containing strains showed similar survival after treatment with UV and ionizing radiation in the presence or absence of oxygen.     

Regulation of the Cell Type-specific dentin sialophosphoprotein gene expression in mouse odontoblasts by a novel transcription repressor and an activator CCAAT-binding factor

Dentin sialophosphoprotein (DSPP) is an extracellular matrix protein that is cleaved into dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) with a highly restricted expression pattern in tooth and bone. Mutations of the DSPP gene are associated with dentin genetic diseases. Regulation of tissue-specific DSPP expression has not been described. To define the molecular basis of this cell-specific expression, we characterized the promoter responsible for the cell-specific expression of the DSPP gene in odontoblasts. Within this region, DNase I footprinting and electrophoretic mobility shift assays delineated one element that contains an inverted CCAAT-binding factor site and a protein-DNA binding site using nuclear extracts from odontoblasts. A series of competitive electrophoretic mobility shift assay analyses showed that the protein-DNA binding core sequence, ACCCCCA, is a novel site sufficient for protein binding. These two protein-DNA binding sequences are conserved at the same proximal position in the mouse, rat, and human DSPP gene promoters and are ubiquitously present in the promoters of other tooth/bone genes. Mutations of the CCAAT-binding factor binding site resulted in a 5-fold decrease in promoter activity, whereas abolishment of the novel protein-DNA binding site increased promoter activity by about 4.6-fold. In contrast to DSPP, expression levels of the novel protein were significantly reduced during odontoblastic differentiation and dentin mineralization. The novel protein was shown to have a molecular mass of 72 kDa. This study shows that expression of the cell type-specific DSPP gene is mediated by the combination of inhibitory and activating mechanisms.     

Molecular markers for ozone stress isolated by suppression subtractive hybridization: specificity of gene expression and identification of a novel stress-regulated gene

Suppression subtractive hybridization was used to identify genes regulated by ozone (100 nmol mol ^{? 1}) in Pisum sativum. One novel gene (named PsUod1) was found. In addition, mRNA levels for four genes (encoding lipid transfer protein, pre‐hevein‐like protein, leucine‐rich repeat protein, and disease‐resistance response protein 230), which previously were shown to be regulated by biotic stress, increased. Finally, mRNA species for two genes (encoding extensin and pathogenesis‐related protein 4A), previously shown to be regulated by ozone in other species, were found to increase in abundance. The ozone‐specificity of the expression of these genes was studied by using UV‐B radiation. PsUod1 and the genes encoding extensin, leucine‐rich repeat protein, and disease‐resistance response protein 230, were differentially regulated when comparing ozone and UV‐B. Moreover, the mRNA levels for extensin, leucine‐rich repeat protein and disease‐resistance response protein 230 all increased under NaCl and aluminium stress and after wounding, whereas the message abundance for PsUod1 was unchanged under these stresses. Thus, in general, ozone caused changes similar to wounding, salt stress and aluminium stress, whereas UV‐B radiation regulated gene expression differently.