We report cloning and tissue expression of transcripts of a fourth fish-specific gene (SPRNB) encoding a prion protein (PrP)-related protein (Shadoo2) from two species, Cyprinus carpio (carp) and Danio rerio (zebrafish), as well as gene or partial transcript sequences identified by in silico searches of public databases from Gasterosteus aculeatus (stickleback), Oryzias latipes (medaka fish) and Salmo salar (salmon). We also present an up-to-date summary of current knowledge of the other three PrP-related genes and transcripts (PrP-like protein, PrP1 and PrP2) encoded exclusively in fish genomes, including some additional transcripts we identified by in silico searches. We compare levels of protein-sequence conservation among the different genes within given fish species, and among given genes in different fish species, and analyse the gene structures and contexts (flanking genes) where genomic sequence is available. These analyses show remarkably high divergences of sequences and expression patterns for proteins in distant fish, such as Fugu and zebrafish, leaving open the question of how varied their functions may be.

Isolation and characterization of both the cDNA and the gene coding for a novel Chlamydomonas reinhardtii protein are here reported. Some peculiar features reported for Chlamydomonas species are recognizable in this mRNA, whose expression resulted to be heat-sensitive. The corresponding nuclear gene exhibits nine introns, all interrupting the coding region and is preceded by a proximal promoter region showing a putative TATA box and a number of sequences fitting heat shock-responsive elements. The deduced protein comprises 169 amino acids and has been called proline-, glycine-, and tyrosine-rich protein (PGYRP), due to the high content of these residues (about 30, 20, and 15%, respectively). The N-terminal region is proline-rich and contains several tandemly repeated hexapeptides sharing the consensus P[Q/P][P/A]GYP, the C-terminal region is rich in glycine, histidine, and lysine, therefore extremely basic; both these domains could be able to interact with other proteins. PGYRP may play a role as adaptor, involved in one or several cellular functions.

Recent findings of new genes in fish related to the prion protein (PrP) gene PRNP, including our recent report of SPRN coding for Shadoo (Sho) protein found also in mammals, raise issues of their function and evolution. Here we report additional novel fish genes found in public databases, including a duplicated SPRN gene, SPRNB, in Fugu, Tetraodon, carp, and zebrafish encoding the Sho2 protein, and we use comparative genomic analysis to analyze the evolutionary relationships and to infer evolutionary trajectories of the complete data set. Phylogenetic footprinting performed on aligned human, mouse, and Fugu SPRN genes to define candidate regulatory promoter regions, detected 16 conserved motifs, three of which are known transcription factor-binding sites for a receptor and transcription factors specific to or associated with expression in brain. This result and other homology-based (VISTA global genomic alignment; protein sequence alignment and phylogenetics) and context-dependent (genomic context; relative gene order and orientation) criteria indicate fish and mammalian SPRN genes are orthologous and suggest a strongly conserved basic function in brain. Whereas tetrapod PRNPs share context with the analogous stPrP-2-coding gene in fish, their sequences are diverged, suggesting that the tetrapod and fish genes are likely to have significantly different functions. Phylogenetic analysis predicts the SPRN/SPRNB duplication occurred before divergence of fish from tetrapods, whereas that of stPrP-1 and stPrP-2 occurred in fish. Whereas Sho appears to have a conserved function in vertebrate brain, PrP seems to have an adaptive role fine-tuned in a lineage-specific fashion. An evolutionary model consistent with our findings and literature knowledge is proposed that has an ancestral prevertebrate SPRN-like gene leading to all vertebrate PrP-related and Sho-related genes. This provides a new framework for exploring the evolution of this unusual family of proteins and for searching for members in other fish branches and intermediate vertebrate groups.

We report evidence from cDNA isolation and expression analysis as well as analyses of genome, expressed sequence tag (EST), cDNA and expression databases for a new gene named SPRN (shadow of prion protein). SPRN comprises two exons, with the open reading frame (ORF) contained within exon 2, and codes for a protein of 130–150 amino acids named Shadoo (Japanese shadow), predicted to be extracellular and GPI-anchored. The SPRN gene was found in fish (zebrafish, Fugu) and mammals (mouse, rat, human). Conservation of order and transcription orientation of two proximal genes between fishes and mammals strongly indicates gene orthology. Sequence comparison shows: a highly conserved N-terminal signal sequence; Arg-rich basic region containing up to six tetrarepeats of consensus XXRG (where X is G, A or S); a hydrophobic region of 20 residues with strong homology to PrP; a less conserved C-terminal domain containing a conserved glycosylation motif; and a C-terminal peptide predicted to be a signal sequence for glycophosphotidylinositol (GPI)-anchor attachment. Fish Shadoos (Sho) show well conserved sequences (identity 54%) over 106 amino acids (zebrafish length), and conservation among the mammalian sequences is very high (identity 81–96%). The fish and mammalian sequences are also well conserved, particularly for zebrafish, to beyond the end of the hydrophobic sequence (identity 41–53%, 78 amino acids, zebrafish length). The overall structure appears closely related to prion proteins (PrPs), although the C-terminal domains of Shos are quite different from those of PrPs, for which conformational changes in mammals are implicated in disease. The structural similarity is particularly interesting given recent reports of three new genes with similarities to PrPs found in Fugu (PrP-like, PrP-461/stPrP-1 and stPrP-2) and other fish, but for which direct evolution to higher vertebrate PrPs is unlikely and for which no other mammalian homologues have been found. Database information indicates expression of SPRN in embryo, brain and retina of mouse and rat, hippocampus of human, and in embryo and retina of zebrafish, and we directly confirmed a strikingly specific expression of the mammalian (human, mouse, rat) transcripts in whole brain. This result together with some common structural features led to the suggestive hypothesis of a possible functional link between mammalian PrP and Sho proteins.

L-Aspartate oxidase is a very particular oxidase which behaves as a fumarate reductase in anaerobic conditions. Its primary and tertiary structures present remarkable similarity with the soluble fumarate reductase (FRD) from Shewanella frigidimarina and the flavin subunit of the membrane-bound fumarate reductase from Escherichia coli and Wolinella succinogenes. This and other extensive similarities are consistent with the idea that a common catalytic mechanism for the reduction of fumarate operates for all members of this enzyme group and that the key residues involved in the substrate binding and catalysis are conserved. This manuscript reports information about the role of these basic residues in L-aspartate oxidase: R290, R386, H244, and H351. By means of site-directed mutagenesis, R290 and R386 are mutated to Leu and H351 and H244 are mutated both to Ala and Ser. H351, H244, and R386 mutants bind substrate analogues with higher dissociation constants and present lower k(cat)/K(m) values in the reduction of fumarate. Therefore, the results indicate that R386, H244, and H351 are important for the binding of the substrate fumarate and may play an important but not essential role in catalysis. R290, on the contrary, is mainly involved in catalysis and not in substrate binding since its mutation abolishes the catalytic activity without lowering the affinity of the enzyme for the substrate. The redox properties of all the mutants are identical to the wild-type. The findings are consistent with a model of L-aspartate oxidase active site based on the hypothesis proposed for the soluble FRD from S. fridimarina.

Isolation and characterization of the cDNA coding for the 216-residue Xenopus laevis prion protein is reported. Existence of this protein in amphibians was suggested by an EST fragment (accession number BG813008), while a conclusive demonstration is presented here. This protein exhibits a higher identity level to avian and turtle prion (more than 44%) than to mammalian prion (about 28%). Although most of the structural motifs common to known prion proteins are conserved in X. laevis, the lack of repeats represents a substantial difference. Other features worth noting are the presence of not perfectly conserved hydrophobic stretch, which is considered the prion signature, as well as the complete absence of histidine residues.

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by bleeding that varies from mild to severe and by complete absence or extremely low levels of plasma and platelet fibrinogen. Although several mutations in the fibrinogen genes associated with dysfibrinogenemia and hypofibrinogenemia have been described, the genetic defects of congenital afibrinogenemia are largely unknown, except for a recently reported 11-kb deletion of the fibrinogen Aalpha-chain gene. Nevertheless, mutation mechanisms other than the deletion of a fibrinogen gene are likely to exist because patients with afibrinogenemia showing no gross alteration within the fibrinogen cluster have been reported. We tested this hypothesis by studying the affected members of two families, one Italian and one Iranian, who had no evidence of large deletions in the fibrinogen genes. Sequencing of the fibrinogen genes in the 2 probands detected 2 different homozygous missense mutations in exons 7 and 8 of the Bbeta-chain gene, leading to amino acid substitutions Leu353Arg and Gly400Asp, respectively. Transient transfection experiments with plasmids expressing wild-type and mutant fibrinogens demonstrated that the presence of either mutation was sufficient to abolish fibrinogen secretion. These findings demonstrated that missense mutations in the Bbeta fibrinogen gene could cause congenital afibrinogenemia by impairing fibrinogen secretion. (Blood. 2000;95:1336-1341)

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by the complete absence of plasma fibrinogen and by a bleeding tendency ranging from mild to moderately severe. Beside a deletion of the almost entire Aα-chain gene, only 2 missense mutations in the C-terminal domain of the Bβ-chain have been very recently described as being associated with afibrinogenemia. We studied a Pakistani patient with unmeasurable plasma levels of functional and immunoreactive fibrinogen. Sequencing of the fibrinogen genes revealed a homozygous G→A transition at position +5 of intron 1 of the γ-chain gene. The predicted mutant fibrinogen γ-chain would contain the signal peptide, followed by a short stretch of aberrant amino acids, preceding a premature stop codon. To demonstrate the causal role of the identified mutation, we prepared expression vectors containing a region of the fibrinogen γ-chain gene spanning from exon 1 to intron 4 and carrying either a G or an A at position +5 of intron 1. Transient transfection of the mutated plasmid in HeLa cells, followed by RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, allowed us to demonstrate the production of an erroneously spliced messenger RNA (mRNA), retaining intron 1, as shown by direct sequencing. A normal splicing occurred in HeLa cells transfected with the wild-type plasmid. This is the first report of a mutation in the fibrinogen γ-chain gene causing afibrinogenemia and indicates that, in addition to the Aα and Bβ-chain genes, the γ-chain gene must also be considered in mutation screening for afibrinogenemia.

Cloning of the cDNA coding for the 270‐residue turtle prion protein is reported. It represents the most remote example thus far described. The entire coding region is comprised in a single exon, while a large intron interrupts the 5′ UTR. The common structural features of the known prion proteins are all conserved in turtle PrP, whose identity degree to mammalian and avian proteins is about 40 and 58%, respectively. The most intriguing feature, unique to the turtle prion, is the presence of an EF‐hand Ca2+ binding motif in the C‐terminal half of the protein.

Isolation and sequencing of bovine and human intron-containing L3 ribosomal protein genes are here reported. They exhibit very similar organisation, both comprising 10 exons and nine introns. A polymorphic locus, involving a 19-bp deletion, was found in intron 6 of the human gene. The frequency of the two alleles has been estimated in 200 haploid genomes. In bovine and human genes intron sequences are rather different, except for limited regions, located in corresponding positions, which show a surprisingly high degree of identity. All these regions contain conserved features defining the box C/D class of small nucleolar RNAs. Demonstration is given that U43 small nucleolar RNA is encoded within the first intron of both bovine and human genes. Single nucleotide sequences, encoding two novel species of small nucleolar RNAs (U82, U83a and U83b), are located in introns 3, 5 and 7. Their expression has been investigated and a possible role of these molecules in 2′-O-ribose methylation of rRNAs is discussed.

The cloning and sequencing of two bovine connexin32 cDNAs are reported. Comparative analysis with known corresponding mammalian cDNA and protein sequences, besides confirming a high degree of similarity among these proteins, allowed us to identify some specific features of the bovine connexin32 gene. The latter include: the presence of a novel exon in the 5' UTR which is alternatively spliced, giving rise to a new mRNA species; the presence of two potential hairpin loops in the 5' and 3' UTR; and the presence of an additional amino acid, glycine235, in the C-terminal domain of the 284 residue protein. Among the common features, the presence of polypyrimidine clusters within the 3' UTR, containing a consensus sequence for a cis-acting element, is noteworthy. Expression of connexin32 mRNAs was analysed in 16 bovine tissues. Transcript analysis suggests the presence, in cattle, of an alternative downstream promoter.

By 16S rDNA sequencing the authors have characterized the coryneform bacteria associated with hyperkeratotic dermatitis (HD) of athymic nude mice isolated from six different outbreaks of the disease in Northern Italy. This analysis has allowed the authors to confirm the classification of the bacteria as Corynebacterium bovis and to develop a 16S rDNA-based polymerase chain reaction (PCR) detection assay. The test was performed directly on the DNA extracted from epidermal swabs. The PCR primers were chosen to match the 16S rDNA sequence fragments which differ most from the other Corynebacterium spp. The test was shown to be both sensitive and specific for C. bovis. Detection of as few as three viable bacterial cells was possible with the use of an oligonucleotide probe in a liquid hybridization assay.

The isolation and sequencing of the complete cDNA coding for a D-aspartate oxidase, as well as the overexpression of the recombinant active enzyme, are reported for the first time. This 2022 bp cDNA, beside the coding portion, comprises a 5' untranslated tract and the whole 3' region including the poly-adenylation signal and the poly(A) tail. The encoded protein comprises 341 amino acids, with the last three residues (-Ser-Lys-Leu) representing a peroxisomal targeting signal 1 (PTS1), hitherto unknown for this protein. The overexpression of recombinant D-aspartate oxidase was achieved in a prokaryotic system, and a soluble and active enzyme was obtained which accounted for about 10% of total bacterial protein. Comparisons with the known cDNAs for mammalian D-amino acid oxidase, another peroxisomal enzyme, are also made. The close structural and functional similarities shared by these enzymes at the protein level are not reflected at the nucleic acid level.

This paper reports the biochemical characterization of the flavoprotein L-aspartate oxidase from Escherichia coli. Modification of a previously published procedure allowed overexpression of the holoenzyme in an unproteolysed form. L-Aspartate oxidase is a monomer of 60 kDa containing 1 mol of noncovalently bound FAD/mol protein. A polarographic and two spectrophotometric coupled assays have been set up to monitor the enzymatic activity continuously. L-Aspartate oxidase was subjected to product inhibition since iminoaspartate, which results from the oxidation of L-aspartate, binds to the enzyme with a dissociation constant (Kd) equal to 1.4 microM. The enzyme binds FAD by a simple second-order process with Kd 0.67 microM. Site-directed mutagenesis of the residues E43, G44, S45, F47 and Y48 located in the putative binding site of the isoallossazinic portion of FAD reduces the affinity for the coenzyme.

L-Aspartate oxidase is a monomeric flavoprotein that catalyzes the first step in the de novo biosynthetic pathway for pyridine nucleotide formation under both aerobic and anaerobic conditions. In spite of the physiological importance of this biosynthesis in particular in facultative aerobic organisms, such as Escherichia coli, little is known about the electron acceptor of reduced L-aspartate oxidase in the absence of oxygen. In this report, evidence is presented which suggests that in vitro fumarate can play such a role. L-Aspartate oxidase binds succinate and fumarate with Kd values of 0.24 mM and 0.22 mM, respectively. A competitive behaviour was observed for these two dicarboxylic acids towards iminoaspartate and sulfite ions. Photoreduction experiments suggest that fumarate and succinate bind at or close to the active site of the molecule. A new fumarate reductase activity of L-aspartate oxidase is reported using benzylviologen or L-aspartate as reductants and fumarate as oxidant. Steady-state kinetics for the oxidase and the fumarate reductase activity of L-aspartate oxidase were obtained using either fumarate or oxygen as electron acceptor and L-aspartate as electron donor. Finally, succinate was identified as the product of the L-aspartate:fumarate oxidoreductase activity using radiolabeled fumarate under anaerobic conditions. The results suggest that fumarate can be a valuable alternative to oxygen as a substrate for L-aspartate oxidase.

A full-length cDNA encoding bovine ribosomal protein L3 was isolated and sequenced. The deduced protein sequence comprises 403 amino acids and shows a high level of identity with the other known mammalian L3 proteins. Southern blot analysis of bovine genomic DNA suggests that the bovine genome contains at least 4 copies of the L3 gene. A single hybridisation band of about 1.3 kb is detectable by Northern blot analysis. Within the amino acid sequence, two potential nuclear targeting sequences were detected: one at the N-terminal end and the other, consisting in a bipartite motif (amino acids 341 to 358), present and not previously noticed also in the other known mammalian L3 proteins. A search on all the available mammalian ribosomal proteins revealed the presence of this bipartite motif in many of these proteins.