Molecular cloning of a CYP1A cDNA from the teleost fish Dicentrarchus labrax

A cDNA encoding for cytochrome P450 1A has been cloned in the marine teleost fish Dicentrarchus labrax. This fish, common in the Mediterranean, was chosen since it is considered a good sentinel species. Moreover, biomarkers of exposure to organic contaminants (such as EROD) are often measured in this species and make it possible to evaluate the quality of waters. For cloning purposes, RNAs were extracted from the liver of benzo[a]pyrene (BaP)-treated animals and used as template in degenerate RT-PCR. The cDNA product was cloned and used for the design of highly stringent primers that were utilized in Rapid Amplification of cDNA Ends (RACE) PCR. The cloned cDNA hybridizes with a 2.7 kb mRNA which is induced by treatment of the fish with BaP, a classical CYP1A inducer. The closest sequences found in data banks belong to fish CYP1A.     

Molecular cloning and nucleotide sequences of cDNA clones of sheep and goat adrenocortical cytochromes P450scc (CYP11A1)

We isolated cDNA clones of the mRNAs for cytochromes P450scc (CYP11A1) from sheep and goat adrenocortices using the RT-PCR method. We determined the complete nucleotide sequences of the coding and 3'-flanking regions of these cDNA clones. The results confirmed the amino terminal sequence of the sheep cytochrome P450scc reported previously [Miyatake et al., Biochim. Biophys. Acta 1215,176-182 (1994)]. On the basis of comparison of the deduced amino acid sequences of various animals and rainbow trout cytochromes P450scc, and other mitochondrial cytochrome P450 isozymes, we discussed the substrate-associated and adreno-ferredoxin-binding region of cytochrome P450scc.     

Adenylosuccinate synthetase genes: molecular cloning and phylogenetic analysis of a highly conserved archaeal gene

Adenylosuccinate synthetase (PurA) catalyzes the first step in the de novo AMP synthesis and has been extensively studied in both Bacteria and Eukarya. We cloned the purA gene from the hyperthermophilic archaeon, Pyrococcus furiosus. The gene appears to be individually transcribed and encodes a protein of 339 amino acids. The amino acid sequence comparison with other archael PurAs found from recent genome analyses indicated that two deletions, one central and the other C-terminal, are a common feature of archaeal PurAs. None of the 21 PurA homologues analyzed from Eukarya and Bacteria exhibited this feature. Amino acid sequences of PurAs in Archaea showed 64% average identities which were significantly higher than the 50% and 55% calculated for Bacteria and Eukarya, respectively. Several residues conserved in PurAs of both Eukarya and Bacteria and shown to be of catalytic importance are missing in the archaeal PurAs. Phylogenetic analysis using PurA as the marker grouped life into 3 domains, hence it was consistent with results derived from 16-18S ribosomal RNA sequences. The topology within the three domains, in general, portrayed the hitherto accepted evolutionary relationship among the organisms utilized. PurA can, thus, serve as an additional marker to evaluate phylogenetic inferences drawn from sequence data from rRNA and other conserved genes. The presence of two unique deletions in both euryarchaeal and crenarchaeal PurAs, but not in those of Bacteria and Eukarya, is a strong evidence confirming the common lineage of these two subdomains of Archaea.     

Molecular cloning and nucleotide sequences of cDNA and gene encoding endo-inulinase from Penicillium purpurogenum

A cDNA and a gene encoding endo-inulinase from Penicillium purpurogenum were isolated, and were cloned for the first time. Two oligonucleotide probes, which were synthesized based on the partial amino acid sequences of the purified endo-inulinase, were used to screen a cDNA library. A 1.7-kb DNA fragment encoding endo-inulinase was isolated and analyzed. A single open reading frame, consisting of 1548-bp, was found to encode a polypeptide that comprised a 25-amino acid signal peptide and 490-amino acid mature protein. All the partial amino acid sequences of the purified enzyme were discovered in the deduced ones. The deduced amino acid sequences of endo-inulinase had similar sequences to those of fructan hydrolases. A 3.5-kb chromosomal DNA fragment encoding endo-inulinase was also isolated and analyzed. The same ORF with cDNA clone as identified. There were no introns in the endo-inulinase gene.     

Intergenic sequences of clustered tRNA genes: new type of genetic marker for phylogenetic studies, with application to the taxonomy of liverworts

We have established a new type of genetic marker in Pellia liverworts that can be used to differentiate between closely related species within this genus. The Pellia intergenic sequences spanning the tandemly repeated nuclear tRNA(Leu) genes were amplified using the PCR technique and, in the case of Pellia neesiana, the tRNA(Leu) tandem region was cloned and sequenced. The length comparison of the intergenic regions allowed for the identification of a number of Pellia species. Additional experiments suggest that this marker can be used to differentiate other species of liverworts and mosses.     

Molecular evolution and host adaptation of Bordetella spp.: phylogenetic analysis using multilocus enzyme electrophoresis and typing with three insertion sequences

A total of 188 Bordetella strains were characterized by the electrophoretic mobilities of 15 metabolic enzymes and the distribution and variation in positions and copy numbers of three insertion sequences (IS). The presence or absence of IS elements within certain lineages was congruent with estimates of overall genetic relationships as revealed by multilocus enzyme electrophoresis. Bordetella pertussis and ovine B. parapertussis each formed separate clusters, while human B. parapertussis was most closely related to IS1001-containing B. bronchiseptica isolates. The results of the analysis provide support for the hypothesis that the population structure of Bordetella is predominantly clonal, with relatively little effective horizontal gene flow. Only a few examples of putative recombinational exchange of an IS element were detected. Based on the results of this study, we tried to reconstruct the evolutionary history of different host-adapted lineages.     

Molecular cloning and sequence analysis of interleukin 16 from nonhuman primates and from the mouse

We analysed the occurrence of anti-neutrophil cytoplasmic antibodies (ANCA) in sera of 191 patients with glomerulonephritis (76 females and 115 males) by the standard indirect immunofluorescence method (IIF). The presence of ANCA was demonstrated in sera of 4.4% (8/181) patients with idiopathic glomerulonephritis (GN) and in 30% (3/10) of patients with rapidly progressive glomerulonephritis (RPGN), as a form of renal limited vasculitis. In the experimental part of our study we analysed the influence of GN ANCA-negative sera on the neutrophil function in vitro and compared with the effect of ANCA-positive sera (titre > or = 4:40) from systemic vasculitis (SV) patients with renal involvement. The activation of neutrophils was established by reactive oxygen species (ROS) production and the ability of superoxide anion to reduce ferrocytochrome c. Among 30 ANCA-negative GN sera 20% (6/30) revealed the ability to activate neutrophils isolated from healthy donor. Remaining ANCA-negative GN sera and all sera from normal healthy individuals (negative control group) did not affect the neutrophil function and did not induce the superoxide anion production. Their effect was similar to the second negative reference system without serum. Only 33% (3/9) of high titre ANCA-positive sera (> or = 1:40) from SV patients were able to activate neutrophils and to produce the superoxide anion with following ferrocytochrome c reduction, but the effect of activation was most powerfully expressed (three times greater than by GN ANCA-negative sera). The remaining ANCA-positive sera and all SV ANCA-negative sera did not affect the neutrophil function in vitro. These experimental data indicate that the presence of ANCA in GN sera is not necessary to induce neutrophil activation in vitro. On the other hand the influence of the SV ANCA-positive sera was most powerful expressed, although only 33% of sera were able to activate neutrophils in vitro. Our results indicate that not always ANCA presence in serum was connected with the ability to neutrophil activation in vitro. It is possible that in ANCA-negative sera other factors were able to activate neutrophils in vitro, but the effect of activation was markedly lower.     

Genotyping of the CYP1A1 and GSTM1 genes in esophageal carcinoma patients with special reference to smoking

OBJECTIVES: Patients with cirrhosis and ascites have high plasma levels of atrial natriuretic peptide (ANP). Pharmacological doses of this hormone usually worsen systemic hemodynamics of cirrhotic patients. We assessed whether ANP influences cardiovascular homeostasis and renal function in patients with compensated cirrhosis at plasma levels comparable to those observed in patients with cirrhosis and ascites. METHODS: Radionuclide angiocardiography was performed in eight compensated cirrhotic patients during placebo (three periods of 15 min each) and ANP infusion (2, 4, and 6 pmol/kg.min for 15 min each), together with appropriate blood and urine sampling, to evaluate left ventricular diastolic, systolic, and stroke volume, heart rate, cardiac output, arterial pressure, peripheral vascular resistance, creatinine clearance, urinary sodium excretion, plasma renin activity, plasma aldosterone, norepinephrine and hematocrit. RESULTS: The infusion increased plasma ANP up to levels (52.03 +/- 2.29 pmol/L) comparable with those observed in 35 patients with ascites (46.42 +/- 1.57 pmol/ L). This increment was associated with significant reductions in left ventricular end diastolic volume, stroke volume, cardiac index (from 3.7 +/- 0.7 to 3.1 +/- 0.5 L/min.m2, p < 0.05) and mean arterial pressure (from 96.7 +/- 6.5 to 88.5 +/- 9.5 mmHg, p < 0.05), while heart rate and hematocrit significantly increased. Peripheral vascular resistance did not change. These hemodynamic effects occurred despite significant increases in plasma renin activity and norepinephrine. ANP also induced increases in creatinine clearance, urinary sodium excretion, and fractional sodium excretion. CONCLUSIONS: Low-dose ANP affected cardiovascular homeostasis and renal sodium handling in compensated cirrhosis, suggesting that this hormone may be involved in the pathophysiology of systemic hemodynamic and renal functional abnormalities of cirrhosis.     

Genetic polymorphism of drug metabolizing enzymes: new mutations in CYP2D6 and CYP2A6 genes in Japanese]

The postnatal development of serotonin (5HT)-immunoreactive axons was studied in the visual cortex of the cerebrum in both normal and microcephalic rats during early postnatal and young adult stages. Severe microcephaly in rat offspring was induced by prenatal exposure to methylazoxymethanol acetate (MAM), an anti-mitotic agent, on day 15 of gestation. From postnatal day 1 (PND 1) to PND 5, fine and short 5HT fibers were irregularly dispersed throughout the occipital cortex in both the control and MAM-treated rats (MAM-rats). A conspicuous aggregation of dot-like 5HT terminals was found in controls, but not in MAM-rats, in a shallow layer of the dorsomedial region of the occipital cortical plate. On PND 7, such an aggregation of 5HT terminals was found in both groups. The density of the aggregation increased up to PND 9, but then decreased gradually, finally becoming unrecognizable at around PND 15 in both groups. MAM-rats, however, always showed hyperaggregation of 5HT terminals when compared with controls on the same PND. The density of 5HT fibers gradually increased, and finally made up a network-like formation at PND 28 in both groups, its pattern was essentially identical to the abnormal distribution of 5HT fibers during the later stage. As a result, the network-like formation of 5HT fibers in the MAM-rats at PND 28 was markedly twisted and somewhat hyperdense. In Nissl-stained preparations from PND 9 to 15, the 5HT terminal aggregation in the control rats was precisely confined to the newly forming layer IV of the visual cortex. In the MAM-rats, on the other hand, the aggregation of 5HT terminals was not associated with a specific cortical layer because of a disarranged cytoarchitecture of the microcephaly.     

Molecular cloning and functional expression of mannitol-1-phosphatase from the apicomplexan parasite Eimeria tenella

A metabolic pathway responsible for the biosynthesis and utilization of mannitol is present in the seven species of Eimeria that infect chickens, but is not in the avian host. Mannitol-1-phosphatase (M1Pase), a key enzyme for mannitol biosynthesis, is a highly substrate-specific phosphatase and, accordingly, represents an attractive chemotherapeutic target. Amino acid sequence of tryptic peptides obtained from biochemically purified Eimeria tenella M1Pase was used to synthesize degenerate oligonucleotide hybridization probes. Using these reagents, a partial genomic clone and full-length cDNA clones have been isolated and characterized. The deduced amino acid sequence of E. tenella M1Pase shows limited overall homology to members of the phosphohistidine family of phosphatases. This limited homology to other histidine phosphatases does, however, include several conserved residues that have been shown to be essential for their catalytic activity. Kinetic parameters of recombinant M1Pase expressed in bacteria are essentially identical to those of the biochemically purified preparation from E. tenella. Moreover, recombinant M1Pase is subject to active site-directed, hydroxylamine-reversible inhibition by the histidine-selective acylating reagent diethyl pyrocarbonate. These results indicate the presence of an essential histidine residue(s) at the M1Pase active site, as predicted for a histidine phosphatase.     

Molecular cloning and characterization of viruses isolated from chimpanzees with pathogenic human immunodeficiency virus type 1 infections

We have previously described the development of AIDS in a chimpanzee (C499) infected with human immunodeficiency virus type 1 (HIV-1) and the subsequent pathogenic HIV-1 infection in another chimpanzee (C455) transfused with blood from C499 (F. J. Novembre et al., J. Virol. 71:4086-4091, 1997). In the present study, two virus isolates were derived from these animals: HIV-1JC from peripheral blood mononuclear cells (PBMC) of C499, and HIV-1NC from plasma of C455. These virus isolates were used to generate two infectious molecular clones, termed HIV-1JC16 and HIV-1NC7 (JC16 and NC7, respectively). Comparative analyses of the sequences of the two clones showed that they were highly interrelated but distinct. Based on heteroduplex mobility assays, JC16 and NC7 appear to represent dominant viruses in the uncloned stock population. Compared with amino acid sequences of the parental viruses HIV-1SF2, HIV-1LAV-1b, and HIV-1NDK, JC16 and NC7 showed a number of differences, including insertions, deletions, and point mutations spread throughout the genome. However, insertion/deletion footprints in several genes of both JC16 and NC7 suggested that recombination between SF2 and LAV-1b could have occurred, possibly contributing to the generation of a pathogenic virus. Comparative in vitro analyses of the molecular clones and the uncloned stocks of HIV-1JC and HIV-1NC revealed that these viruses had strikingly similar replicative abilities in mitogen-stimulated PBMC and in macrophages. Compared to the SF2 and LAV-1b isolates of HIV-1, HIV-1JC and HIV-1NC isolates were more similar to LAV-1b with respect to the ability to replicate in mitogen-stimulated PBMC and macrophages. These viruses should prove to be useful in mapping determinants of pathogenesis.     

Molecular cloning and analysis of a lipase gene from Pseudomonas fluorescens No. 33

The gene encoding an extracellular lipase from Pseudomonas fluorescens No. 33 was cloned and sequenced. A single open reading frame consisting of 1,428 nucleotides that encoded a mature protein of 476 amino acids was recognized. Sequence analysis showed that the deduced molecular weight of 50,209 agreed with the molecular weight of the purified lipase as measured by SDS-PAGE and the lipase lacked a signal peptide. The presence of a repeating motif, GXXGXDXXX, suggested that the lipase might be exported and secreted via a system that involves the ATP-binding cassette protein.