Cloning and sequencing of a 2,5-dichlorohydroquinone reductive dehalogenase gene whose product is involved in degradation of gamma-hexachlorocyclohexane by Sphingomonas paucimobilis

Spinal metabotropic glutamate receptors (mGluR) have been implicated in hyperalgesia after injury. The purpose of this study was to examine the effects of intrathecal (IT) mGluR antagonists on mechanical hyperalgesia in a rat model of human postoperative pain. The hindpaw withdrawal threshold to punctate stimulation using von Frey filaments and the response frequency to a nonpunctate stimulus applied directly to the wound were also measured. The effects of 1T (+)-alpha-methyl-carboxyphenylglycine ([+]-MCPG), (S)-carboxyphenylglycine ([S]-4-CPG), (RS)-alphacyclopropyl-4-phosphonophenylglycine ([RS]-CPPG) and L-2-amino-3-phosphonopropionic acid (L-AP3) on incision-induced mechanical hyperalgesia were examined. The withdrawal thresholds to punctate stimuli were not different from vehicle treatment after the IT administration of (+)-MCPG (100, 500 nmol), (S)4CPG (30, 100 nmol), (RS)-CPPG (100, 500 nmol), or L-AP3 (1, 30, 100 nmol). None of the IT mGluR antagonists decreased the response frequency to the nonpunctate stimulus. The largest dose of (+)-MCPG produced sufficient receptor antagonism because spontaneous nociceptive behaviors caused by the IT administration of a mGluR agonist were reduced. IMPLICATIONS: Spinal metabotropic glutamate receptors antagonists, antinociceptive in some models of persistent pain, are not necessary for the maintenance of mechanical hyperalgesia in this rat model, which suggests that blockade of spinal metabotropic glutamate receptors may not be useful for the treatment of pain after surgery.     

Cloning of a Sphingomonas paucimobilis SYK-6 gene encoding a novel oxygenase that cleaves lignin-related biphenyl and characterization of the enzyme

Sphingomonas paucimobilis SYK-6 transforms 2,2'-dihydroxy-3,3'-dimethoxy-5,5'-dicarboxybiphenyl (DDVA), a lignin-related biphenyl compound, to 5-carboxyvanillic acid via 2,2',3-trihydroxy-3'-methoxy-5,5'-dicarboxybiphenyl (OH-DDVA) as an intermediate (15). The ring fission of OH-DDVA is an essential step in the DDVA degradative pathway. A 15-kb EcoRI fragment isolated from the cosmid library complemented the growth deficiency of a mutant on OH-DDVA. Subcloning and deletion analysis showed that a 1.4-kb DNA fragment included the gene responsible for the ring fission of OH-DDVA. An open reading frame encoding 334 amino acids was identified and designated ligZ. The deduced amino acid sequence of LigZ had 18 to 21% identity with the class III extradiol dioxygenase family, including the beta subunit (LigB) of protocatechuate 4,5-dioxygenase of SYK-6 (Y. Noda, S. Nishikawa, K.-I. Shiozuka, H. Kadokura, H. Nakajima, K. Yano, Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki, J. Bacteriol. 172:2704-2709, 1990), catechol 2,3-dioxygenase I (MpcI) of Alcaligenes eutrophus JMP222 (M. Kabisch and P. Fortnagel, Nucleic Acids Res. 18:3405-3406, 1990), the catalytic subunit of the meta-cleavage enzyme (CarBb) for 2'-aminobiphenyl-2,3-diol from Pseudomonas sp. strain CA10 (S. I. Sato, N. Ouchiyama, T. Kimura, H. Nojiri, H. Yamane, and T. Omori, J. Bacteriol. 179:4841-4849, 1997), and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) of Escherichia coli (E. L. Spence, M. Kawamukai, J. Sanvoisin, H. Braven, and T. D. H. Bugg, J. Bacteriol. 178:5249-5256, 1996). The ring fission product formed from OH-DDVA by LigZ developed a yellow color with an absorption maximum at 455 nm, suggesting meta cleavage. Thus, LigZ was concluded to be a ring cleavage extradiol dioxygenase. LigZ activity was detected only for OH-DDVA and 2,2',3,3'-tetrahydroxy-5,5'-dicarboxybiphenyl and was dependent on the ferrous ion.     

Cloning, sequencing, and expression of the Zymomonas mobilis phosphoglycerate mutase gene (pgm) in Escherichia coli

Phosphoglycerate mutase is an essential glycolytic enzyme for Zymomonas mobilis, catalyzing the reversible interconversion of 3-phosphoglycerate and 2-phosphoglycerate. The pgm gene encoding this enzyme was cloned on a 5.2-kbp DNA fragment and expressed in Escherichia coli. Recombinants were identified by using antibodies directed against purified Z. mobilis phosphoglycerate mutase. The pgm gene contains a canonical ribosome-binding site, a biased pattern of codon usage, a long upstream untranslated region, and four promoters which share sequence homology. Interestingly, adhA and a D-specific 2-hydroxyacid dehydrogenase were found on the same DNA fragment and appear to form a cluster of genes which function in central metabolism. The translated sequence for Z. mobilis pgm was in full agreement with the 40 N-terminal amino acid residues determined by protein sequencing. The primary structure of the translated sequence is highly conserved (52 to 60% identity with other phosphoglycerate mutases) and also shares extensive homology with bisphosphoglycerate mutases (51 to 59% identity). Since Southern blots indicated the presence of only a single copy of pgm in the Z. mobilis chromosome, it is likely that the cloned pgm gene functions to provide both activities. Z. mobilis phosphoglycerate mutase is unusual in that it lacks the flexible tail and lysines at the carboxy terminus which are present in the enzyme isolated from all other organisms examined.     

Cloning and sequencing of the LEU2 homologue gene of Schwanniomyces occidentalis

A gene that complements the leu2 mutation of Saccharomyces cerevisiae has been cloned from Schwanniomyces occidentalis. The gene codes for a protein of 379 amino acids. As expected for a Schwanniomyces gene, it has a high AT content, which is also reflected in the codon usage. The sequence homology with other known leu2 complementing genes is low.     

Cloning and expression of the major porin protein gene opcP of Burkholderia (formerly Pseudomonas) cepacia in Escherichia coli

OBJECTIVES: We sought to assess the effects of aging on the endothelial physiology of a group of Chinese adults. BACKGROUND: Several studies have documented an association between aging and progressive arterial endothelial dysfunction in white subjects. We hypothesized that age-related endothelial dysfunction, an important event in atherosclerosis, might be less marked in southern Chinese subjects, in whom the prevalence of coronary heart disease is only approximately 20% of that in industrialized countries. METHODS: We studied endothelial function in 76 healthy adults aged 16 to 70 years: 38 Chinese from a village of 3,000 people in southern China and 38 white subjects from Sydney, Australia. In each ethnic group, there were 19 younger persons (16 to 40 years) and 19 older adults (55 to 70 years). None had evidence of diabetes, hypertension or clinical vascular disease or had ever been regular cigarette smokers. With the use of high resolution external vascular ultrasound, brachial artery diameter was measured at rest, after flow increase (causing endothelium-dependent dilation) and after sublingual nitroglycerin (an endothelium-independent dilator). RESULTS: Endothelium-dependent dilation was similar in young Chinese (mean +/- SD 8.3 +/- 2.5%), young whites (7.9 +/- 2.0%) and older Chinese (6.8 +/- 2.9%), but it was significantly impaired in older whites (1.8 +/- 2.5%, p < 0.001 by analysis of variance). On multivariate analysis, older age was associated with impaired endothelium-dependent dilation (p < 0.001) (independent of the effects of serum cholesterol, gender and vessel size) in the white but not in the Chinese subjects (p = 0.83). Nitroglycerin-induced dilation was not significantly different with aging in either ethnic group. CONCLUSIONS: Endothelium-dependent dilation is similar in the arteries of healthy young Chinese and white adults. With older age, however, Chinese subjects are less susceptible to impaired endothelial function.     

Cloning and sequencing of the para-type sodium channel gene from susceptible and kdr-resistant German cockroaches (Blattella germanica) and house fly (Musca domestica)

This paper presents the results of viscosity determinations on aqueous solutions of bovine serum albumin (BSA) at a wide range of concentrations and at temperatures ranging from 5 degrees C to 45 degrees C. On the basis of these measurements a general formula connecting the relative viscosity eta r with temperature T and concentration c of the dissolved proteins was established: [formula: see text] The quantities alpha, beta, B, D and delta E are described in the text below. A simple substantiation of the formula was also given. This relation gives immediately the Mooney approximation and allows the prediction of the values of the parameter S and a self-crowding factor K in this approximation. By applying an asymptotic form of the formula such rheological quantities as the intrinsic viscosity and Huggins coefficient were calculated.     

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.     

Cloning and sequencing of the URA3 gene of Kluyveromyces marxianus CBS 6556

More than 250 mutations have been detected in the cystic fibrosis (CF) transmembrane regulator (CFTR) gene, most of which are single point mutations or small deletions or insertions of a few nucleotides. Here we report the first large deletion identified in the CFTR gene, which involves 50 kb in two stretches of DNA: one of 10 kb from exon 4 to exon 7, and another of 40 kb, spanning exons 11 to 18. The deletion has been detected via uniparental inheritance of CFTR microsatellite alleles (IVS17BTA and IVS17BCA) in 3 independent CF families. Clinical status of the 3 CF patients, of which two have the delta F508 mutation as the other CF allele, suggests that this mutation is responsible for a severe clinical phenotype, indistinguishable from homozygous delta F508 patients. The deletion detected here suggests that other large, but less complex molecular defects could also exist in the CFTR gene.     

Cloning, sequencing and expression of the CAMP factor gene of Streptococcus uberis

The gene coding for the CAMP factor from a strain of Streptococcus uberis (ATCC 9927) was cloned in Escherichia coli. Chromosomal DNA from Streptococcus uberis was used to construct a gene library in plasmid pTZ18R and six CAMP-reaction positive clones were obtained from a total of 10,000 transformants. One clone, pJLD21, was subcloned and the CAMP factor gene was located in a 3.2 kb BamHI fragment. The nucleotide sequence of Streptococcus uberis CAMP factor gene was determined and the deduced amino acid sequence is highly homologous to the corresponding Streptococcus agalactiae protein. Immunoblot analysis revealed that the recombinant strain pJLD21 expressed a protein with a molecular weight of 28 000. Antibodies raised against purified Streptococcus uberis CAMP factor cross-reacted with Streptococcus agalactiae protein B.     

Cloning, sequencing and overexpression of the leucine dehydrogenase gene from Bacillus cereus

OBJECTIVE: To define the significance of prepubertal diabetes duration in the development of diabetic microvascular complications in adolescents. RESEARCH DESIGN AND METHODS: Study A compares complications in 38 prepubertal (PreP) and 140 pubertal (Pub) subjects of the same age (10-14 years) and diabetes duration (3-12 years) to determine if the absence of puberty itself confers a lower risk of complications. Study B examines the importance of prepubertal and pubertal diabetes duration in 193 older adolescents (ages 15-22 years) with prepubertal onset of diabetes. Retinopathy status was assessed using stereoscopic fundus photography of seven fields per eye. Albumin excretion rate (AER) was assessed by three consecutive overnight urine collections, using a polyclonal radioimmunoassay. RESULTS: In study A, there were no significant differences between the PreP and Pub groups for retinopathy (27 vs. 29%, P = 0.8) or differences in elevated AER (17 vs. 31%, P = 0.1). In study B, longer prepubertal diabetes duration improved the prediction for retinopathy over postpubertal duration alone (P < 0.0005). No relationship with duration was found for elevated AER (> 7.5, > 15, and > 30 micrograms/min). CONCLUSIONS: Prepubertal subjects with diabetes did not have less retinopathy or elevated albumin excretion compared with pubertal subjects of the same age. Prepubertal diabetes duration is significantly related to the presence of retinopathy in adolescents.     

Cloning and sequencing of potato virus Y (Hungarian isolate) genomic RNA

Through multiple logistic regression an epidemiological study was undertaken of the following factors: age, gender, socio-economic status, dental care, toothbrushing, chewing gum, snacking, fluoride, and of their influence on the development of tooth decay. The factors are analysed individually and globally (global model). An initial model was constructed, establishing the interactions, and developing a final model. Risk factors shown to be involved were: low social class status, lack of dental care in the previous 12 months, absence of toothbrushing, and belonging to the age group 9-12 years old. An interaction was established between the following variables: socio-economic status and toothbrushing, and dental care and age.     

Cloning and sequencing of the gene for the Thiobacillus ferrooxidans ATCC33020 glutamate synthase (GOGAT) small subunit and complementation of an Escherichia coli gltD mutant

A recombinant plasmid which contains the gltD gene coding for the glutamate synthase (GOGAT) small subunit was isolated from a Thiobacillus ferrooxidans ATCC33020 gene bank by complementation of an Escherichia coli gltD mutant. The sequence of gltD was determined. The deduced amino acid sequence shows strong similarity to the two other prokaryote gltD sequences available, namely those of E. coli and A. brasilense (53% and 45% identity, respectively). A cosmid containing the gltBD region was isolated from a T. ferrooxidans cosmid gene bank, but was unable to complement an E. coli gltB mutant.     

Cloning and sequencing of the gene encoding a 31-kilodalton antigen of Haemophilus somnus

Immunoblots using bovine antibody against Haemophilus somnus as the primary antibody consistently identified 31-, 40- and 78-kDa proteins in Sarkosyl-insoluble extracts of H. somnus. A genomic library of H. somnus 8025 DNA was constructed in plasmid pUC19, and 45 recombinants expressed proteins which were recognized by bovine antiserum in Western blots (immunoblots). Ten of the recombinants expressing a 31-kDa protein caused the lysis of bovine erythrocytes. Restriction endonuclease mapping indicated that the hemolytic recombinants shared an approximately 1.7-kb BglII fragment. Southern blot analysis using the BglII fragment as a probe revealed homology among the recombinants and the presence of an identically sized BglII fragment in the chromosome of all H. somnus isolates tested. Sequence analysis indicated the presence of an 822-bp open reading frame within the 1.7-kb BglII fragment. Deletion of this open reading frame resulted in the loss of hemolytic activity and protein expression in recombinant Escherichia coli, suggesting the possible role of the 31-kDa protein as a hemolysin. An amino acid sequence deduced from the DNA sequence shared homology with outer membrane protein A of E. coli, Salmonella typhimurium, and Shigella dysenteriae, with P6 of Haemophilus influenzae, and with PIII of Neisseria gonorrhoeae. An amino acid analysis of the recombinant 31-kDa protein agreed with the amino acid composition deduced from the DNA sequence.     

Cloning and sequencing of a full-length sea bream (Sparus aurata) beta-actin cDNA

The pathogenesis of human African trypanosomiasis (HAT) has been the object of considerable research interest but has remained incompletely understood. The importance of cytokines in the pathophysiology of this protozoan infection is now widely recognized, but the full spectrum of cytokines involved has yet to be determined. In the present investigation we compared the plasma concentrations of TNF-alpha and IL-10 in normal African controls and patients suffering from advanced meningocephalic (late-stage) Trypanosomiasis brucei (T.b.) gambiense infections, before and after treatment with the arsenical trypanocide melarsoprol. We found that patients with late-stage T. b. gambiense exhibit chronically elevated circulating levels of both of these cytokines, and that these levels quickly decline following melarsoprol treatment. These findings confirm that TNF-alpha is involved in the immunopathogenesis of late-stage African trypanosomiasis and suggest that IL-10 may also play an important regulatory role in this disease.     

Cloning, sequencing and expression of the gene encoding elongation factor P in the amino-acid producer Brevibacterium lactofermentum (Corynebacterium glutamicum ATCC 13869)

The Brevibacterium lactofermentum EF-P gene, encoding the elongation factor protein P, was cloned and sequenced. According to DNA sequence analysis of this gene, the B. lactofermentum EF-P protein consists of 187 amino acids with a calculated molecular weight of 20,584. Southern hybridization of an internal fragment of the EF-P gene from B. lactofermentum with chromosomal DNAs from different microorganisms reveals that it is a unique gene product in B. lactofermentum and Corynebacterium glutamicum. The EF-P gene was expressed in E. coli using the T7 expression system and the calculated molecular weight of the expressed protein was 23,000. Disruption experiments using an internal fragment of the EF-P gene or a disrupted EF-P gene in suicide plasmids always failed, suggesting that the gene is needed for cell viability.     

Cloning and sequencing of the gene encoding a novel lysine-specific cysteine proteinase (Lys-gingipain) in Porphyromonas gingivalis: structural relationship with the arginine-specific cysteine proteinase (Arg-gingipain)

Lys-gingipain (KGP), so termed due to its peptide cleavage specificity for lysine residues, is a cysteine proteinase produced by the Gram-negative anaerobic bacterium Porphyromonas gingivalis. Mixed oligonucleotide primers designed from the NH2-terminal sequence of the purified enzyme were used to clone the KGP-encoding gene (kgp) from the organism. The nucleotide sequence of kgp had a 5,169-bp open reading frame encoding 1,723 amino acids with a calculated molecular mass of 218 kDa. As the extracellular mature enzyme had an apparent molecular mass of 51 kDa in gels, the precursor of KGP was found to comprise at least four domains, the signal peptide, the NH2-terminal prodomain, the mature proteinase domain, and the COOH-terminal hemagglutinin domain, and to be proteolytically processed during its transport. Importantly, the COOH-terminal region contained three direct repeats of two different amino acid sequences, LKWD(or E)AP and YTYTVYRDGTKI, and the subdomains located between the two repeats exhibited strong similarity to those of Arg-gingipain (RGP), another major cysteine proteinase produced by the organism and having cleavage specificity for arginine residues, although the arrangement of the subdomains was not necessarily identical in the two enzymes. Since the KGP activity was greatly decreased in RGP-deficient mutants and since the most probable site of the propeptide cleavage was present in the homologous sequence highly susceptible to proteolysis by RGP, the precursor of KGP is likely to be processed by RGP to form the mature enzyme.     

Cloning, sequencing, mapping and hyperexpression of the ribC gene coding for riboflavin synthase of Escherichia coli

The upper layers of mammalian epidermis contain citrulline-containing proteins formed by enzymatic deimination of arginine residues. To study the role of protein deimination in epidermal differentiation, we identified deiminated proteins extracted from human epidermis. Major deiminated proteins were identified as partially degraded keratin K1, while those from keratin K10 and a highly heterogeneous mixture of deiminated filaggrin isomers were detected as minor components. Deiminated keratins were recovered in a fraction enriched with keratins from the cornified layers. The subsequent immunohistochemical study showed that deiminated proteins were localized mainly in the lowermost cornified layer, but not in the granular layer. These data suggested that partially degraded/disulfide-cross-linked keratin K1 was preferentially deiminated during the terminal stages of epidermal differentiation. We therefore speculated that the protein deimination might influence the interaction of basic K1 with its acidic partner K10, pre-existent K5/K14 networks or keratin-associated protein filaggrin.     

Cloning, sequencing and expression of the acylneuraminate lyase gene from Clostridium perfringens A99

The acylneuraminate lyase gene from Clostridium perfringens A99 was cloned on a 3.3 kb HindIII DNA fragment identified by screening the chromosomal DNA of this species by hybridization with an oligonucleotide probe that had been deduced from the N-terminal amino acid sequence of the purified protein, and another probe directed against a region that is conserved in the acylneuraminate lyase gene of Escherichia coli and in the putative gene of Clostridium tertium. After cloning, three of the recombinant clones expressed lyase activity above the background of the endogenous enzyme of the E. coli host. The sequenced part of the cloned fragment contains the complete acylneuraminate lyase gene (ORF2) of 864 bp that encodes 288 amino acids with a calculated molecular weight of 32.3 kDa. The lyase structural gene follows a noncoding region with an inverted repeat and a ribosome binding site. Upstream from this regulatory region another open reading frame (ORF1) was detected. The 3'-terminus of the lyase structural gene is followed by a further ORF (ORF3). A high homology was found between the amino acid sequences of the sialate lyases from Clostridium perfringens and Haemophilus influenzae (75% identical amino acids) or Trichomonas vaginalis (69% identical amino acids), respectively, whereas the similarity to the gene from E. coli is low (38% identical amino acids). Based on our new sequence data, the 'large' sialidase gene and the lyase gene of C. perfringens are not arranged next to each other on the chromosome of this species.