The vascular endothelial-cadherin promoter directs endothelial-specific expression in transgenic mice.

Vascular endothelial-cadherin (VE-cadherin) is a calcium-dependent adhesive molecule, exclusively and constitutively expressed in endothelial cells. Analysis of the VE-cadherin promoter fused to a reporter gene in bovine aortic endothelial cells showed three major functional regions. The proximal region alone (-139, +24) promoted nonspecific transcription; the addition of the (-289, -140) and (-2226, -1190) domains abolished transcription in fibroblasts while expression in endothelial cells remained unchanged, suggesting that fragments (-2226, +24) and longer contain the full endogenous promoter activity. To study the transcriptional specificity of the promoter region in vivo, we generated transgenic mice carrying the chimeric construct containing the (-2486, +24) region. The promoter directed reporter expression in all examined organs of adult transgenic mice. During embryonic development, transgene expression was detected at the early steps of vasculogenesis. Later, the expression persisted during development of the vascular system and was restricted to the endothelial layer of the vessels. Together, these data provide evidence for specific regulatory regions within the VE-cadherin promoter. Furthermore, the identification of DNA sequences restricting gene expression to the endothelium has many potential applications for the development of animal models of cardiovascular or angiogenic diseases or for the delivery of therapeutic molecules.

Requirement of a GT box (Sp1 site) and two Ets binding sites for vascular endothelial cadherin gene transcription.

Vascular endothelial cadherin (VE cadherin) gene encodes a Ca2+-dependent cell adhesion molecule required for the organization of interendothelial junctions. This gene is exclusively and constitutively expressed in endothelial cells. Previous data with transgenic mice revealed that the transcriptional regulatory elements present within a -2486/+24 DNA fragment of mouse VE cadherin gene mimic the tissue-specific activity of the endogenous promoter. In this study, we analyzed elements implicated in the function of the proximal regulatory region. Electrophoretic mobility shift assay identified a GT-rich sequence (positions -49/-39) interacting with factors related to the Sp1 family. Point mutations abolished the binding of nuclear proteins in vitro and drastically diminished the activity of the promoter in transient transfection assay. Supershift assays with antibodies against proteins of the Sp1 family revealed that Sp1 and Sp3 interact with this region of the VE cadherin promoter. Furthermore, two GGAA motifs, located at positions -93/-90 and -109/-106, were shown to interact with nuclear factors. Site-directed mutagenesis of these sequences demonstrated that these Ets binding sites are essential for promoter activity. In vitro binding assays in the presence of various antisera suggest that Erg is one of the proteins interacting with the -109/-106 site.

Genomic structure and chromosomal mapping of the mouse VE-cadherin gene (Cdh5).

Vascular endothelial cadherin (VE-cadherin) is located strictly at endothelial junctions and appears to be a major adhesive component of cell to cell contacts. Genomic clones spanning 36 kb and encompassing the mouse VE-cadherin gene have been isolated and characterized. The gene is composed of 12 exons that exhibit conventional vertebrate splicing. The first exon is entirely untranslated, and both exons 2 and 12 contain untranslated regions. A single major transcriptional start site was identified and located 75 bases upstream of the translation initiation codon in the cDNA sequence. The proximal 5'-flanking domain lacks consensus TATA and CAAT boxes at the usual positions. Exon-intron boundaries are similar to those of other cadherin genes, with some exceptions that may have a functional significance in VE-cadherin behavior. The VE-cadherin gene (locus Cdh5) maps to mouse chromosome 8, where it colocalizes with E-cadherin (locus Cdh1), P-cadherin (locus Cdh3), and M-cadherin (locus Cdh14) genes, suggesting that it might be part of a larger cluster of cadherin sequences.

The human beta fibrinogen promoter contains a hepatocyte nuclear factor 1-dependent interleukin-6-responsive element.

Acute-phase reactants are liver proteins whose synthesis is positively or negatively regulated during inflammation. The main mediators of this phenomenon are glucocorticoids and interleukin-6 (IL-6), a pleiotropic cytokine that also controls hematopoiesis. Functional analysis of several acute-phase reactant promoter regions has identified two major DNA motifs used by IL-6-regulated genes. The first one corresponds to a CTGG(G/A)AA sequence, and the other is a binding site for members of the C/EBP family of nuclear proteins. We have previously shown that the human beta fibrinogen (beta Fg) promoter contains an IL-6-responsive region, located between bp -150 and -67 (P. Huber, M. Laurent, and J. Dalmon, J. Biol. Chem. 265:5695-5701, 1990). In this study, using DNase I footprinting, mobility shift assays, and mutagenesis, we demonstrate that at least three subdomains of this region are necessary to observe a full response to IL-6. The most distal contains a CTGGGAA motif, and its mutation inhibits IL-6 stimulation. Another, which is able to interact with several distinct nuclear proteins, among them members of the C/EBP family, is dispensable for IL-6 induction but plays an important role in the constitutive expression of beta Fg. Finally, a proximal hepatocyte nuclear factor 1 binding site, already described as the major determinant of beta Fg tissue-specific expression, is also required for IL-6 stimulation. These results indicate a complex interplay between nuclear proteins within the beta Fg IL-6-responsive region and suggest a tight functional coupling between the tissue-specific and inducible elements.

Human beta-fibrinogen gene expression. Upstream sequences involved in its tissue specific expression and its dexamethasone and interleukin 6 stimulation.

The synthesis of fibrinogen in the liver is drastically enhanced during the inflammatory process. Two factors are involved: glucocorticoids and the hepatocyte stimulating factor which is identical with interleukin 6 (Il6), also called interferon beta 2. The function of the 5'-flanking region of the human beta-fibrinogen (beta-Fg) gene has been studied by deletion analysis with the chloramphenicol acetyltransferase (CAT) gene as a transient expression vector. In this analysis, a fragment containing 150 base pairs (bp) upstream from the cap site is sufficient to drive expression of the CAT gene in the hepatoma cells HepG2, but not in HeLa cells. The beta-Fg gene is induced by dexamethasone and Il6 in HepG2. We identify a domain located between -2900 and -1500 bp upstream from the transcription start point involved in dexamethasone sensibility. This distal regulatory region can confer hormone inductibility to a heterologous promoter and exert its effect in either orientation. The sequence located between -150 and -82 bp upstream from the transcription start point is responsive for the Il6-stimulated expression. This 68-bp sequence contains probably all the cis-acting Il6-responsive element of the human beta-Fg gene.

Characterization of the 5'-flanking region for the human fibrinogen beta gene.

To identify the possible regulatory sequences in the genetic expression of fibrinogen, a human genomic DNA library raised in lambda EMBL 4 phage was screened using cDNA probes coding for the A alpha, B beta and gamma chains of human fibrinogen. The entire fibrinogen locus was characterized and its organization analysed by means of hybridization and restriction mapping. Among the clones identified, a single recombinant lambda phage contained the beta gene and its 5'- and 3'-flanking regions. A 1.5 kb fragment of the immediate 5'-flanking region was sequenced and S1 mapping experiments revealed three transcription start points. Comparison of this sequence with that previously reported for the same region upstream from the human gamma gene revealed no significant homology which suggests that the potential promoting sequences of these genes are different. In contrast, comparison of the 5'-flanking regions of human and rat beta genes revealed a 142 bp sequence of 80% homology situated 16 bp upstream from the human beta gene. This highly conserved region may well represents a potential candidate for a regulatory sequence of the human beta gene.

5-Methylcytosine in Chlorelle pyrenoidosa DNAs.

The presence of 5-methylcytosine in Chlorella pyrenoidosa (strain 211/8b) DNA's has been investigated by means of paper chromatography and thermal chromatography on hydroxyapatite. It has been shown that nuclear DNA contains 3.5 mol% 5-methylcytosine whereas no significant amount of this base can be detected in chloroplast DNA. The thermal chromatography of nuclear DNA labelled from [6-3H]- or [Me-14C] methionine lead us to conclude that the 5-methylcytosine content is directly proportional to the G + C content of the various DNA fractions. The existence of methylated sequences in DNA is postulated and the biological function of the 5-methylcytosine is discussed.

[Physico-chemical determination of the ploidy of the unicellular alga, Chlorella pyrenoidosa (strain 211/8b) (author's transl)]. / Determination physico-chimique de la ploidie chez l'algue unicellulaire Chlorella pyrenoidosa (souche 211/8b)

The ploidy of the unicellular green alga Chlorella pyrenoidosa (strain 211/8b) has been determined by means of renaturation kinetics. The nuclear DNA is made up from fast, intermediate and slow renaturing sequences, which represent respectively about 5, 15 and 80% of the DNA. These observations are consistent with the findings in other eukaryotic nuclear DNAs. Nevertheless, the relative importance of the repeated sequences is much lower than that observed in Chlamydomonas reinhardi [16] and in higher plants [18-20], but slightly higher than that obtained in Chlorella vulgaris [17]. The kinetic complexity of the main fraction of the Cl. pyrenoidosa nuclear DNA is found to be 2.94 - 10-10 daltons (mean value of five independant experiments) assuming value of 2.1 - 10-8 daltons for Cl. pyrenoidosa chloroplastic DNA. When compared with the analytical complexity of this fraction (80% of the nuclear DNA analytical complexity, that is 2.02 - 10-10 daltons), one can assume that the slow renaturing fraction of the nuclear DNA is constituted by a unique nucleotide sequence. This result thus suggests that Cl. pyrenoidosa (strain 211/8b) is an haploid organism. The possible existence of an haploid genome in the nuclei of the algae from Chlorella genus and the apparent absence of sexuality might explain the high discrepancy observed in the G + C content of the Chlorella nuclear DNAs.

The chloroplastic DNA of Chlorella pyrenoidosa (Emerson strain): heterogeneity and complexity.

The heterogeneity and the complexity of Emerson strain Chlorella pyrenoidosa chloroplastic DNA have been investigated by means of thermal denaturation and renaturation kinetics, and the results have been compared with those of the strain 211/8b of the same alga. The thermal denaturation properties are very close to those of the other strain: the Tm of 65 degrees C in 0.1 standard saline citrate, the maximal hyperchromicity of 41%, and the dispersion coefficent delta 2/3 of 6.65 degrees C. The first derivated curves of the melting profiles show also five components. Denatured chloroplastic DNA renatures rapidly. Two fractions are found; their kinetic complexities have been estimated: 1.5 times 10(7) daltons for the fast renaturing fraction; 2x 10(8) daltons for the low d (G + C) content of the chloroplastic DNA: 1.24 times 10(8) daltons). The unique nucleotide sequence is present in about 19 copies per chloroplastic genome. This report confirms the homogeneity of the chloroplastic genome of algae.