EDA is a facultative type III homology of human fibronectin encoded by an alternative spliced exon. The EDA+ and EDA- mRNA forms show a cell type specific distribution with their relative proportion varying during development, aging and oncogenic transformation. We have previously demonstrated that an 81 bp nucleotide sequence within the exon itself is essential for differential RNA processing. Fine mapping of cis acting elements within this region has been carried out to identify possible target sites for the modulation of alternative splicing. There are at least two short nucleotide sequences involved. Element A (GAAGAAGA) is a positive modulator for the recognition of the exon, its deletion results in constitutive exclusion of the EDA exon. Element B (CAAGG) is a negative modulator for exon recognition, its deletion results in constitutive inclusion of the EDA exon. This bipartite structure of the splicing enhancer is a novel feature of the mammalian exons.

We present the characterization of two overlapping human transferrin genomic clones isolated from a liver DNA library. The two clones represent a total length of 24 kilobase pairs and code for 70% of the protein. The

Abstract.Fibronectin isoforms are generated by the alternative splicing of a primary transcript derived from a single gene. In rat at least three regions of the molecule are involved: EIIIA, EIIIB, and V. This study investigated the splicing patterns of these regions during development and aging, by means of ribonuclease protection analysis. Between fetal and adult rat, the extent of inclusion of the EIIIA and/or EIIIB region in fibronectin mRNA varied according to the type of tissue analyzed; but the inclusion of the V region, and in particular the V25 alternative variant, was significantly higher in all fetal than in adult tissues. These data suggest a crucial role of the V25 variant, possibly related to its interaction with the c~4~1 integrin receptor during development. On the other hand, during aging, the only significant change observed in the splicing pattern was a decrease in the EIIIA variant in brain. The high inclusion levels of the EIIIA and EIIIB regions in young adult brain suggest that these segments may play an important role in differentiated brain tissue. The decreasing levels of inclusion of the EIIIA segment in brain fibronectin mRNA during aging may be an age-related marker with functional consequences. CELL-to-cell and cell-to-substrate interactions play a fundamental role in cell behavior, division, and differentiation. Among the extracellular components involved in these events, fibronectin (FN) ~ is probably one of the most important and widely distributed. Fibronectin is a dimeric high molecular weight glycoprotein found in blood, lymph, and tissue fluids, as well as in association with basement membranes, connective tissue matrices and the extracellular matrix of many cells (23, 37).The primary structure of FN has been described at both protein (human and bovine FN) and cDNA (chicken, rat, and human FN) levels (4, 13-15, 21, 26, 32, 33, 40, 41, 43, 45). The FN molecule is composed of three different kinds of repeated sequence, known as types I, II and III with a characteristic modular structure (21,33,40,41,43). These repeats are assembled into a series of structural domains, each having a distinct binding activity toward collagen, sulfated glycosaminoglycans, fibrin, and the cell surface receptors collectively termed integrins (19,36,49).FN molecules are a mixture of several protein types that differ both in their primary structure, and in their posttranslational modifications. All of the sequence variations are produced by the alternative processing of a common mRNA precursor (pre-mRNA) transcribed from a single gene and 1. Abbreviation used in this paper: FN, fibronectin. differentially spliced in the various cell types (1,18,30,40,41,45,50).Three sites of alternative splicing have so far been described: ED-A, ED-B, and IIICS in human FN and the corresponding sites (EIIIA, EIIIB, and V) in rat FN. EIIIA and EIIIB share a common gene structure and, in both cases, alternative splicing leads to the insertion of an additional exon coding for a type III homology domain. The V region in ...

Vasostatins (VS) are vasoinhibitory peptides derived from the N-terminal domain of chromogranin A, a secretory protein present in the electron-dense granules of many neuroendocrine cells. In this work we describe a method for the production in Escherichia coli of large amounts of recombinant vasostatins, corresponding to chromogranin A residues 1-78 (VS-I), and 1 -115 (VS-2), and the use of these materials for structure characterisation. The masses of both products were close to the expected values, by SDS/ PAGE and mass spectrometry analysis. However, their hydrodynamic behaviours in size-exclusion chromatography corresponded to that of proteins with a larger size. SDSPACE analysis of VS-1 and VS-2 after cross-linking with disuccinimidyl suberate indicated that both polypeptides form dimers. VS-2 was almost entirely dimeric at > 4 pM, but rapidly converted to monomer after dilution to 70 nM. The rapid dimer-monomer transition of VS-2 after dilution could be part of a mechanism for regulating its activity and localising its action. Immunological studies of VS-1 have shown that residues 37-70 constitute a highly antigenic region characterised by an abundance of linear epitopes efficiently mimicked by synthetic peptides. The recombinant products and the immunological reagents developed in this work could be valuable tools for further investigating the structure and the function of chromogranin A and its fragments.Keywords: chromogranin; vasostatin ; recombinant DNA ; monoclonal antibody; antigenic region.Chromogranin A is an acidic protein belonging to a family of regulated secretory proteins present in the electron-dense granules of many endocrine and neuroendocrine tissues [I -31. Human chromogranin A is a hydrophilic protein of 439 amino acids, characterised by several post-translational modifications, including glycosylation, sulfation and phosphorylation [ 1, 4, 51. Chromogranin A has been suggested to be a low-affinity highcapacity Ca' + -binding protein involved in hormone packaging within secretory granules and in the regulation of secretory granule biogenesis and function [6, 71. Moreover, chromogranin A has been proposed to represent a precursor of biologically active peptides that are released together with various hormones [8]. Accordingly, chromogranin A contains a high number of dibasic sites thought to be important for tissue-specific proteolytic processing and biological activity [9-121. For instance, residues 248 -293 were found to be similar to pancreastatin, a pancreatic peptide that inhibits insulin secretion [I 31, whereas residues 124-143, called chromostatin, inhibit secretion of catechol- amines from chromaffin cells (141. Stimulated retrogradely perfused bovine adrenal medullae release the chromogranin A fragments corresponding to amino acids 1-76 and 1-113. These fragments, named vasostatin I and 11, respectively, were found to be endowed with vasoinhibitory activity on human blood vessels [lS, 161. Studies aimed at investigating the structure and the function of chromogranin A and its ...

The mature hen avidin encoded by a synthetic cDNA was expressed in Escherichia coli in an insoluble form. After resolubilization, renaturation and purification, a recovery of about 20 mg/l cell culture was obtained. ELISA assays indicated no apparent differences in biotin binding between the natural and recombinant avidins. In addition, an acidic avidin mutant, bearing the substitutions Lys3→Glu, Lys9→ Glu, Arg26→Asp and Arg124→Leu of four exposed basic residues, was produced. The protein, expressed and renatured as wild-type avidin, showed unaltered biotin-binding activity. The acidic pI (Ϸ5.5) and lack of aggregation of the mutant allowed easy electrophoretic analysis under non-denaturing conditions of the protein alone and of its complexes with biotin, biotinylated transferrin or peroxidase. Analysis of the sera from sensitized subjects revealed that the avidin mutant has altered antigenicity. Both recombinant avidins were crystallized and the three-dimensional structures solved by molecular replacement and refined to 0.22 nm resolution. The three-dimensional structures of the two recombinant molecules, in the absence of biotin and of glycosylation, are fully comparable with those of the natural hen avidin previously reported.Keywords : avidin; crystal structure ; mutagenesis ; recombinant protein.Avidin, a minor constituent of egg white of reptiles, amphibia and birds, is a glycosylated and positively charged protein which can bind up to four molecules of vitamin H, D-biotin [1]. The interaction with biotin is non-covalent but extremely tight, the dissociation constant of about 1 fM being about 10 3 Ϫ10 6 times higher than that of a typical antigen-antibody interaction. The cDNA-derived and protein-derived sequences [2,3] and the crystallographic structures of avidin [4Ϫ6] are known. Functional avidin is a tetramer of identical subunits and each subunit is folded into an eight-stranded anti-parallel β-barrel displaying up-and-down topology. The biotin-binding site is located in the core of the barrel, and is built by residues of the barrel itself and partly by a loop of an adjacent subunit. The interest in avidin derives mainly from several applications based on its rapid and almost irreversible binding to biotin and to practically any biotin-based molecular construct [7,8]. Avidin is used in vivo for the targeting of solid tumors [9] in applications where proteinsurface properties are determinants for proper biodistribution, serum clearance and immunological response [10]. The production of recombinant avidin and its mutated forms could increase the already remarkable versatility of this protein and may improve its properties for in vivo use.