Cloning and nucleotide sequence of amidase gene from Pseudomonas putida

The peptidergic signal substance thyrotropin-releasing hormone (TRH) is inactivated by the TRH-degrading ectoenzyme (TRH-DE), a peptidase that exhibits an extraordinary high degree of substrate specificity and other unusual characteristics. There is no other ectopeptidase known capable of degrading this tripeptideamide, and vice versa, TRH is the only known substrate of this unique enzyme. Thus, studies on this enzyme may reveal new aspects on the function of the TRH signaling system. After succeeding in purifying this enzyme to homogeneity and cloning the cDNA encoding rat TRH-DE, molecular tools became available to study the expression of this enzyme by Northern blot analysis and in situ hybridization histochemistry. The stringent and tissue-specific regulation of the adenohypophyseal TRH-DE by estradiol and thyroid hormones strongly suggests that this enzyme may act as a regulatory element modulating pituitary hormone secretion. In brain, the expression of TRH-DE is not influenced by peripheral hormones but the distinct distribution pattern, and the high activities support the concept that in this tissue TRH-DE may act as a terminator of TRH signals.     

Helicobacter pylori hspA-hspB heat-shock gene cluster: nucleotide sequence, expression, putative function and immunogenicity

All Helicobacter pylori isolates synthesize a 54 kDa immunodominant protein that was reported to be associated with the nickel-dependent urease of H. pylori. This protein was recently recognized as a homologue of the heat-shock protein of the GroEL class. The gene encoding the GroEL-like protein of H. pylori (HspB) was cloned (pILL689) and was shown to belong to a bicistronic operon including the hspA and hspB genes. In Escherichia coli, the constitutive expression of the hspA and hspB genes was initiated from a promoter located within an IS5 insertion element that mapped upstream to the two open reading frames (ORFs). IS5 was absent from the H. pylori genome, and was thus acquired during the cosmid cloning process. hspA and hspB encoded polypeptides of 118 and 545 amino acid residues, corresponding to calculated molecular masses of 13.0 and 58.2 kDa, respectively. Amino acid sequence comparison studies revealed that, although H. pylori HspA and HspB proteins were highly similar to their bacterial homologues, the H. pylori HspA featured a striking motif at the C-terminus. This unique motif consists of a series of cysteine and histidine residues resembling a nickel-binding domain, which is not present in any of the other bacterial GroES homologues so far characterized. When the pILL689 recombinant plasmid was introduced together with the H. pylori urease gene cluster (pILL763) into an E. coli host strain, an increase of urease activity was observed. This suggested a close interaction between the HspA and HspB proteins and the urease enzyme, and a possible role for HspA in the chelation of nickel ions. The genes encoding each of the HspA and HspB polypeptides were cloned, expressed independently as proteins fused to the maltose-binding protein (MBP) and purified in large scale. The MBP-HspA and MBP-HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H. pylori-infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic.     

COFFEE: an objective function for multiple sequence alignments

MOTIVATION: In order to increase the accuracy of multiple sequence alignments, we designed a new strategy for optimizing multiple sequence alignments by genetic algorithm. We named it COFFEE (Consistency based Objective Function For alignmEnt Evaluation). The COFFEE score reflects the level of consistency between a multiple sequence alignment and a library containing pairwise alignments of the same sequences. RESULTS: We show that multiple sequence alignments can be optimized for their COFFEE score with the genetic algorithm package SAGA. The COFFEE function is tested on 11 test cases made of structural alignments extracted from 3D_ali. These alignments are compared to those produced using five alternative methods. Results indicate that COFFEE outperforms the other methods when the level of identity between the sequences is low. Accuracy is evaluated by comparison with the structural alignments used as references. We also show that the COFFEE score can be used as a reliability index on multiple sequence alignments. Finally, we show that given a library of structure-based pairwise sequence alignments extracted from FSSP, SAGA can produce high-quality multiple sequence alignments. The main advantage of COFFEE is its flexibility. With COFFEE, any method suitable for making pairwise alignments can be extended to making multiple alignments. AVAILABILITY: The package is available along with the test cases through the WWW: http://www. ebi.ac.uk/cedric CONTACT: cedric.notredame@ebi.ac.uk     

Misleading local sequence alignments: implications for comparative protein modelling

Although it is well known that significant sequence similarity between proteins is reflected at the structural level, it is commonly assumed that any misaligned regions, as judged by the correct structure based alignment, are those where the local sequence identity is lower than the global. Recent studies have shown that this is not always the case and there can exist short stretches of high local identity which is not reflected in the structure based alignment. An analysis is presented of 290 pairs of homologous proteins with a view to quantifying the occurrence of these misleading local sequence alignments (MLSAs). It is found that such MLSAs are likely if the global sequence identity is less than 40% and can occur even when it is greater than 60%. The results have implications for automated homology modelling and also for the inference of function made by comparison.     

Molecular cloning and nucleotide sequence of the protyrosinase gene, melO, from Aspergillus oryzae and expression of the gene in yeast cells

The coding region of the protyrosinase gene, melO, from Aspergillus oryzae occupies 1671 base pairs of the genomic DNA and is separated into two exons by one intron. The full-length cDNA of the melO gene was cloned. Analysis of the 1617 base pairs nucleotide sequence revealed a single open reading frame coding 539 amino acid residues. The cDNA has been expressed in yeast cells. The predicted protein product derived from the melO gene is identified by Western blotting and activity determination. The predicted amino acid sequence of the gene product was compared with that of Neurospora crassa tyrosinase. A coupled pair of three histidine residues in the tyrosinase was assumed to correspond to Cu(II) ligands in the homologous tyrosinases from Streptomyces glaucescens and Homo sapiens.     

Molecular cloning and nucleotide sequence of the genomic DNA for 1,2-alpha-D-mannosidase gene, msdC from Penicillium citrinum

A gene encoding 1,2-alpha-D-mannosidase (EC 3.2.1.113) was cloned from Penicillium citrinum genomic DNA using the polymerase chain reaction (PCR). The coding region of the gene, msdC, occupied 1737 bp and was separated into four exons by three introns. The predicted protein consisted of 511 amino acid residues with M(r) 56,569. Penicillium enzyme had a hydrophobic signal peptide at the N-terminal region as did mammalian membrane-bound alpha-mannosidases, but in this case a proteolytic cleavage occurred at Lys-35-Ser-36 to remove the signal sequence during cell growth. Parts of amino acid sequences were similar to those of mammalian Golgi alpha-mannosidase IA and IB, but the sequence around the aspartic acid residue which interacted with 1-deoxymannojirimycin (Yoshida et al. (1994) Biochem. J. 303, 97-103) was unique in Penicillium enzyme.     

Allelic difference in the nucleotide sequence of the Eta-1/Op gene transcript

A 40-year-old man developed general malaise, dizziness and progressive headache 1 week after acute haemorrhagic conjunctivitis. Bizarre behaviour, confusion and adversive seizures occurred later. MRI showed haemorrhagic infarct in the bilateral basal ganglia and thalamus and abnormal signal density in the internal cerebral veins. Bilateral carotid angiography showed no filling of superior sagittal sinus and internal cerebral veins. With MRI, cerebral venous thrombosis can be suspected and diagnosed earlier than before, obviating any invasive investigation.     

Molecular cloning and nucleotide sequence of an endo-1,5-alpha-L-arabinase gene from Bacillus subtilis

The nucleotide sequence of the gene encoding an endo-1,5-alpha-L-arabinase (protopectinase C) of Bacillus subtilis was determined by sequencing fragments amplified by the cassette-ligation-mediated PCR (CLM-PCR). The gene covering the start and stop codon was amplified by PCR with two specific primers, which were designed from the sequence data determined by CLM-PCR. An approximately 1.5-kb amplification product was cloned into the vector pUC119, forming a plasmid termed pPPC. An ORF that encodes the arabinase composed of 324 amino acids including a 33-amino-acid signal peptide was assigned. Comparison of the deduced amino acid sequence of the enzyme with that of an Aspergillus niger endoarabinase showed 37% identity in a 207-amino-acid overlap. The optimal nucleotide sequence for catabolite repression of B. subtilis was found upstream of the structural gene. In a culture of Escherichia coli DH5alpha cells harboring pPPC, no arabinase activity was detected, either intracellularly or extracellularly, suggesting that the B. subtilis promotor is not functional in this transformant. In B. subtilis IFO 3134 strain, production of protopectinase C was repressed by readily metabolizable carbohydrates. In contrast, productivity (total enzyme activity/bacterial growth) of the enzyme was increased about fourfold in the presence of 0.75 M potassium phosphate in the culture medium. The phosphate anion seemed to be involved in the stimulation of protopectinase C production in this stain.     

Evolution of the cytochrome P450 superfamily: sequence alignments and pharmacogenetics

The evolution of the cytochrome P450 (CYP) superfamily is described, with particular reference to major events in the development of biological forms during geological time. It is noted that the currently accepted timescale for the elaboration of the P450 phylogenetic tree exhibits close parallels with the evolution of terrestrial biota. Indeed, the present human P450 complement of xenobiotic-metabolizing enzymes may have originated from coevolutionary 'warfare' between plants and animals during the Devonian period about 400 million years ago. A number of key correspondences between the evolution of P450 system and the course of biological development over time, point to a mechanistic molecular biology of evolution which is consistent with a steady increase in atmospheric oxygenation beginning over 2000 million years ago, whereas dietary changes during more recent geological time may provide one possible explanation for certain species differences in metabolism. Alignment between P450 protein sequences within the same family or subfamily, together with across-family comparisons, aid the rationalization of drug metabolism specificities for different P450 isoforms, and can assist in an understanding of genetic polymorphisms in P450-mediated oxidations at the molecular level. Moreover, the variation in P450 regulatory mechanisms and inducibilities between different mammalian species are likely to have important implications for current procedures of chemical safety evaluation, which rely on pure genetic strains of laboratory bred rodents for the testing of compounds destined for human exposure.     

Genomic organization and complete nucleotide sequence of the human PWP2 gene on chromosome 21

The human PWP2 gene is the human homologue of the yeast periodic tryptophan protein 2 (PWP2) gene and is a member of the gene family that contains tryptophan-aspartate (WD) repeats. Genomic sequencing revealed that the human PWP2 gene consists of 21 exons spanning approximately 24 kb and locates just between the two genes EHOC-1 and KNP-I and distal to a NotI site of LJ104 (D21S1460) on chromosome 21q22.3. Analysis of the 5'-flanking DNA sequence revealed that the upstream region of the PWP2 gene is associated with a CpG island containing the NotI site of LJ104. Since PWP2 is considered to be a candidate for genetic disorders mapped in the 21q22.3 region, the information including nucleotide sequence and genomic organization of the PWP2 gene should be invaluable for the mutation analysis of the corresponding genetic disorders.     

Cloning and nucleotide sequence analysis of a gene encoding an OXA-derived beta-lactamase in Acinetobacter baumannii

A clinical strain of Acinetobacter baumannii (strain Ab41) that was resistant to all beta-lactam antibiotics tested except ceftazidime, ceftriaxone, ceftizoxime, and imipenem produced three beta-lactamases: a presumptive chromosomal cephalosporinase, a TEM-1-like beta-lactamase (pI 5.4), and a novel OXA-derived beta-lactamase named OXA-21 (pI 7.0). The gene encoding OXA-21 was located in an integron. The nucleotide sequence showed three mutations compared with the sequence of OXA-3, with two being silent; the nonsilent mutation generated a substitution of Ile-217 to Met.     

The transcriptional activator HlyU of Vibrio cholerae: nucleotide sequence and role in virulence gene expression

HlyU upregulates expression of the haemolysin, HlyA, of Vibrio cholerae. DNA sequence analysis indicates that HlyU is an 11.9 kDa protein containing a putative helix-turn-helix motif and belonging to a family of small regulatory proteins, including NoIR (Rhizobium meliloti), SmtB (Synechococcus PCC 7942) and ArsR (plasmids R773, Escherichia coli; pI258, Staphylococcus aureus; and pSX267, Staphylococcus xylosus). An hlyU mutant was constructed by insertional inactivation, and found to be deficient in the production of both the haemolysin and a 28 kDa secreted protein. The mutant was assessed for virulence in the infant mouse cholera model, revealing a 100-fold increase in the LD50. This suggests that HlyU promotes expression of virulence determinant(s) in vivo.     

Mouse liver nicotinamide N-methyltransferase: cDNA cloning, expression, and nucleotide sequence polymorphisms

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide and structurally related compounds. We cloned mouse liver NNMT cDNA to make it possible to test the hypothesis that large differences among strains in levels of hepatic NNMT activity might be associated with strain-dependent variation in NNMT amino acid sequence. Mouse liver NNMT cDNA was 1015 nucleotides in length with a 792 nucleotide open reading frame (ORF) that was 83% identical to the nucleotide sequence of the human liver NNMT cDNA ORF. The mouse liver cDNA encoded a 264 amino acid protein with a calculated Mr value of 29.6 kDa. NNMT cDNA ORF sequences were then determined in five inbred strains of mice with very different levels of hepatic NNMT enzymatic activity. Although multiple differences among strains in nucleotide sequence were observed, none altered encoded amino acids. cDNA sequences for C57BL/6J and C3H/HeJ mice, prototypic strains with "high" and "low" levels of hepatic NNMT activity, respectively, were then expressed in COS-1 cells. Both expression constructs yielded comparable levels of enzyme activity, and biochemical properties of the expressed enzyme, including apparent Km values for substrates and IC50 values for inhibition by N1-methylnicotinamide, were very similar to those of mouse liver NNMT. Growth and development experiments were then conducted, which demonstrated that, although at 8 weeks of age average hepatic NNMT activity in C57BL/6J mice was 5-fold higher than that in C3H/HeJ mice, activities in the two strains were comparable by 30 weeks of age--indicating strain-dependent variation in the developmental expression of NNMT in mouse liver. These observations will serve to focus future studies of strain-dependent differences in murine hepatic NNMT on the regulation of the enzyme activity during growth and development.