Transcription factors of the bHLH-PAS protein family are important regulators of developmental processes such as neurogenesis and tracheal development in invertebrates. Recently a bHLH-PAS protein, named trachealess (trl) was identified as a master regulator of tracheogenesis. Hypoxia-inducible factor, HIF-1 alpha, is a vertebrate relative of trl which is likely to be involved in growth of blood vessels by the induction of vascular endothelial growth factor (VEGF) in response to hypoxia. In the present study we describe mRNA cloning and mRNA expression pattern of mouse HIF-related factor (HRF), a novel close relative of HIF-1 alpha which is expressed most prominently in brain capillary endothelial cells and other blood vessels as well as in bronchial epithelium in the embryo and the adult. In addition, smooth muscle cells of the uterus, neurons, brown adipose tissue and various epithelial tissues express HRF mRNA as well. High expression levels of HRF mRNA in embryonic choroid plexus and kidney glomeruli, places where VEGF is highly expressed, suggest a role of this factor in VEGF gene activation similar to that of HIF-1 alpha. Given the similarity between morphogenesis of the tracheal system and the vertebrate vascular system, the expression pattern of HRF in the vasculature and the bronchial tree raises the possibility that this family of transcription factors may be involved in tubulogenesis.

Vascular endothelial growth factor (VEGF)--also known as vascular permeability factor--has been implicated in the regulation of blood vessel formation, i.e., vasculogenesis and angiogenesis. High amounts of VEGF mRNA and protein have been detected during embryonic and tumor angiogenesis, but it remained unclear whether the level of VEGF correlated with the extent of vascularization in a given organ or tissue. We examined the role of VEGF and the high affinity, signal-transducing VEGF receptor-2 (flk-1) in the avian embryo. In a gain of function transgene-like approach the retroviral expression vector RCAS was used to increase the level of quail VEGF during critical periods of avian limb bud growth and morphogenesis. In contrast to basic fibroblast growth factor, which recently was demonstrated to induce morphogenetic alterations when overexpressed in this system, overexpression of VEGF in the limb bud exclusively resulted in hypervascularization as reflected by an increase in vascular density. However, cartilage expressing the construct was not vascularized prematurely. Thus hypervascularization was probably due to the augmentation of the VEGF signaling mechanism in a permissive environment. In addition to hypervascularization, vascular permeability was dramatically increased, leading to local and in some cases to general edema. This is the first indication of a link between the functions of VEGF as a vascular growth factor and as a permeability factor. VEGF receptor-2 (flk-1) was found to be upregulated only in those areas where VEGF was overexpressed. This implies a positive feedback system of the VEGF receptor on its own synthesis and would provide a basis for a paracrine system in which ligand concentration is critical for the extent of tissue vascularization. Our results show that the VEGF/VEGF-receptor system is specific and sufficient for the formation of new blood vessels. They also have implications for somatic gene therapy of diseases which are characterized by a lack of blood vessels such as chronic ischemic diseases of heart and brain.

We have previously shown that IS5 contains two genes encoded on opposite DNA strands within the same stretch of DNA. Here we present evidence that a third gene and its promoter are present on IS5. The newly discovered gene, ins5C, is contained within the longest gene of ISS, ins5A, but encoded by the complementary DNA strand. The three genes comprise a total of 519 codons present on the 1195-bp element. The arrangement of these genes represents a coding structure of unprecedented compactness. Key words: transposable element/insertion element/IS5 genes/IS5 promoters/IS5 Results Detection and localization of the promoter p5C To find transcription start signals on IS5 in addition to the ones previously identified, IS5 DNA fragments were inserted into the promoter-search vector-plasmid pK01 (McKenney et al., 1981;Rak et al., 1982) which carries the galactokinase structural gene without a promoter. A 970-bp BstEII-HindIII fragment, derived from plasmid pFDI (pFRI in Rak et al., 1982; see Figure 1), containing IS5: 1-543 and also 427 bp of X DNA (X:36 895-37 322;Sanger et al., 1982;Kroger and Hobom, 1982), was digested with restriction endonuclease HaeIII and the resulting fragments were isolated. The different HaeIII fragments were ligated into the SmaI site of pKOl and the ligation products were transformed into strain N100 (galK, recA). Transformants containing hybrid plasmids which carried the HaeIII fragment in both possible orientations were obtained for all fragments and screened on Introduction Insertion (IS) elements form a group of relatively small (-. 800-1500 bp) DNA sequences found in the genomes of bacteria and in the DNA of their phages and plasmids. Their properties have recently been reviewed by lida et al. (1983). Evidence has accumulated that IS elements not only contain specific sites recognized by proteins mediating their transposition, but also code for proteins required for this event. The frequency of transposition appears to be influenced by the physiological state of the host cell (Arber et al., 1979(Arber et al., , 1981 Arber and lida, 1982), but the ability to regulate transposition functions seems to be a property of the IS element itself (Beck et al., 1980;Biek and Roth, 1980; see also lida et al., 1983). In the case of ISIO it has been proposed that this regulation may be implemented by IS-specific transcripts which inhibit the translation of a message for transposase (Simons and Kleckner, 1983). In the case of IS50 it has been suggested that regulation occurs at a post-translational level, involving an IS-encoded protein (Isberg et al., 1982). No