Molecular cloning of the cDNA coding for Xenopus laevis prion protein.

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.

Probing the active site of L-aspartate oxidase by site-directed mutagenesis: role of basic residues in fumarate reduction.

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.

Afibrinogenemia: first identification of a splicing mutation in the fibrinogen gamma chain gene leading to a major gamma chain truncation.

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 Aalpha-chain gene, only 2 missense mutations in the C-terminal domain of the Bbeta-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 gamma-chain gene. The predicted mutant fibrinogen gamma-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 gamma-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 gamma-chain gene causing afibrinogenemia and indicates that, in addition to the Aalpha and Bbeta-chain genes, the gamma-chain gene must also be considered in mutation screening for afibrinogenemia.

cDNA cloning of turtle prion protein.

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 Ca(2+) binding motif in the C-terminal half of the protein.

Missense mutations in the human beta fibrinogen gene cause congenital afibrinogenemia by impairing fibrinogen secretion.

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)

The intron-containing L3 ribosomal protein gene (RPL3): sequence analysis and identification of U43 and of two novel intronic small nucleolar RNAs.

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.

A new exon in the 5' untranslated region of the connexin32 gene.

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.

Analysis of the 16S rRNA gene sequence of the coryneform bacterium associated with hyperkeratotic dermatitis of athymic nude mice and development of a PCR-based detection assay.

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.

cDNA cloning and expression of the flavoprotein D-aspartate oxidase from bovine kidney cortex.

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 polyadenylation 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.

L-aspartate oxidase from Escherichia coli. I. Characterization of coenzyme binding and product inhibition.

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 from Escherichia coli. II. Interaction with C4 dicarboxylic acids and identification of a novel L-aspartate: fumarate oxidoreductase activity.

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.

cDNA sequence for bovine ribosomal protein L3 carrying a bipartite nuclear targeting motif, identified also in many other ribosomal proteins.

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.

The primary structure of the flavoprotein D-aspartate oxidase from beef kidney.

The complete primary structure of the peroxisomal flavoenzyme D-aspartate oxidase from beef kidney has been determined by analyses of the peptides obtained through fragmentation of the carboxymethylated protein with trypsin, CNBr, heptafluorobutyric acid/CNBr and Staphylococcus aureus V8 protease. The protein consists of a single polypeptide of 338 residues, accounting for a M(r) of 37,305 for the apoprotein. A form of the enzyme lacking Lys-338 and therefore ending with Pro-337 has been detected. The N-terminal residue is blocked. Seven cysteines and no disulfide bridges are present. Residue 228 can be either Ile or Val. Thus, D-aspartate oxidase presents two types of heterogeneity in the polypeptide chain in addition to the one already described concerning the possible content of FAD or 6-hydroxyflavin adenine dinucleotide. Comparison of the primary structure of D-aspartate oxidase with other known sequences reveals that D-aspartate oxidase is homologous with D-amino acid oxidase (another flavo-oxidase) and does not present significant sequence similarities with any other protein, including flavoenzymes.

Assignment of the human connexin 32 gene (GJB1) to band Xq13.

The chromosomal localization of the human gene coding for connexin 32 (GJB1) was determined by in situ suppression hybridization (ISSH). The results allowed assignment of the gene to band Xq13, thus refining previous localization data obtained by means of somatic cell hybrid analysis.

A biotin-avidin sandwich enzyme-linked immunosorbent assay of growth hormone in bovine plasma.

Purified pituitary bovine growth hormone (bGH) has been used to develop a homologous sandwich enzyme-linked immunosorbent assay in which affinity-purified antibodies are immobilized on microtiter plates. Bovine GH bound to specific antibody is then revealed with a second anti-bGH antibody labeled with biotin and peroxidase-conjugated avidin. The method requires only 48 h, including the coating step, and has a sensitivity as low as 0.25 ng/ml of bGH. Statistical analyses (test of parallelism, cross-reactivity among bGH and GH of various species and bovine prolactin, recovery test, within- and between-assay variation, comparison with radioimmunoassay) confirm the high specificity and reproducibility of the method.

C5a fragment of bovine complement. Purification, bioassays, amino-acid sequence and other structural studies.

C5a and des-Arg-C5a have been purified from bovine serum in milligram amounts. The progress of the purification was followed by measuring the chemotactic activity of the complement fragments. The two polypeptides induce activation of neutrophil-oriented locomotion and secretion with very similar dose/response effects. After preparing a rabbit antiserum to bovine C5a/des-Arg-C5a, a competitive enzyme-linked immunosorbent assay (ELISA) was set up for the detection of C5a from 5 ng/mol to 1 microgram/ml. The complete primary structure of bovine C5a, which consists of 74 amino acids, has been determined by sequence analysis of the tryptic peptides, aligned by peptides derived from a chymotryptic digest, and by partially sequencing the intact molecule. Bovine C5a has a sequence homology of 78% and 70% with porcine and human C5a, respectively, reacts with an antiserum to porcine C5a and is recognized by cell surface receptors on human neutrophils. Finally, the secondary structure of bovine C5a was investigated by circular dicroic spectroscopy and predicted from the amino acid sequence. A comparison of the content and distribution of alpha-helical and/or hydropathic regions, suggests that the three-dimensional structure of C5a might be modeled from the known crystal structure of the homologous C3a molecule.

Fasciola hepatica: increase of glycogen phosphorylase activity due to prostaglandins.

Both prostaglandin E1 (PGE1) and prostaglandin F2 alpha (PGF2 alpha) stimulate the glycogen phosphorylase (EC 2.4.1.1.) activity of Fasciola hepatica. Whole or sliced parasites were incubated with PGE1 (2.8 X 10(-7) and 2.8 X 10(-5) M) and PGF2 alpha (2.1 X 10(-7) and 2.1 X 10(-5) M) and enzyme activity was measured in homogenates prepared immediately following the incubation. No substantially different effect was noted between the two assayed doses of prostaglandins. Prostaglandins appeared to be less effective in sliced parasites.