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Molecular Cloning (molecular + cloning)
Selected AbstractsMolecular Cloning and Characterization of CD9 cDNA from Sheep and Cashmere GoatREPRODUCTION IN DOMESTIC ANIMALS, Issue 3 2010WJ Xing Contents CD9 is a glycoprotein of the transmembrane 4 superfamily (TM4SF) and is involved in various cellular processes. Some CD9 cDNA have been cloned in mammals and certain fish genera in recent years, but goat and sheep counterparts of cattle, human and mouse have not been identified. To facilitate the studies, we cloned the cDNA encoding for CD9 of cashmere goat (Capra hircus) and sheep (Ovis aries), and expressed sheep CD9 in Escherichia coli cells. Structural analysis indicated for both goat and sheep that a 1123 bp cDNA spanned an open reading frame of 681 bp which predicted a protein of 226 amino acids with a typical TM4SF structure, including four highly conserved transmembrane domains, two extracellular domains and a CCG motif, which is a hallmark of the TM4SF. The predicted amino acid sequences were highly homologous to those of cattle, mouse and human CD9. Molecular phylogenetic analysis based on CD9 cDNA sequences indicated that goat and sheep CD9 were closely related to CD9 of cattle, which is in agreement with their morphological taxonomy. [source] Molecular Cloning and Characterization of a Gene Encoding a 13.1 kDa Antigenic Protein of Naegleria fowleriTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 6 2001HO-JOON SHIN ABSTRACT. An antigen-related gene was cloned from a cDNA expression library of Naegleria fowleri by immunoscreening with sera obtained from mice that were either immunized with an amoebic lysate or infected with trophozoites. The coding nucleotide sequence of the cloned gene consisted of 357 bases that were translated into 119 amino acids. This gene was designated as nfal. The predicted amino acid sequence of Nfal protein has two potential glycosylation and three potential phosphorylation sites, and its predicted secondary structure consists of four helices and three corners. The deduced amino acid sequence of Nfal protein shares 43% identity with the myohemerythrin (myoHr) protein from a marine annelid, Nereis diversicolor, including 100% identity in conserved regions and iron-binding residues. A phylogenetic tree constructed from amino acid sequences placed the N. fowleri Nfal protein outside of a cluster of myoHr proteins from eight invertebrates. A purified recombinant protein that migrated as a 13.1 kDa species in SDS-PAGE was produced. This recombinant protein exhibited a strong immunoreactivity with infected, immune, and anti-Nfal sera. In addition. an anti-Nfal serum reacted with an amoeba lysate in immunoblotting analysis. The present nfal gene encoding the myoHr-like protein is the first myoHr gene cloned from protozoa, and the Nfal antigen may be useful in diagnostic studies. [source] Developmental anatomy of reeler mutant mouseDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2009Yu Katsuyama The reeler mouse is one of the most famous spontaneously occurring mutants in the research field of neuroscience, and this mutant has been used as a model animal to understand mammalian brain development. The classical observations emphasized that laminar structures of the reeler brain are highly disrupted. Molecular cloning of Reelin, the gene responsible for reeler mutant provided insights into biochemistry of Reelin signal, and some models had been proposed to explain the function of Reelin signal in brain development. However, recent reports of reeler found that non-laminated structures in the central nervous system are also affected by the mutation, making function of Reelin signal more controversial. In this review, we summarized reported morphological and histological abnormalities throughout the central nervous system of the reeler comparing to those of the normal mouse. Based on this overview of the reeler abnormalities, we discuss possible function of Reelin signal in the neuronal migration and other morphological events in mouse development. [source] Molecular cloning and sequence analysis of an ascidian egg ,-N-acetylhexosaminidase with a potential role in fertilizationDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2003Ryo Koyanagi ,-N-Acetylhexosaminidase, which is found almost ubiquitously in sperm of invertebrates and vertebrates, supposedly mediates a carbohydrate-based transient sperm,egg coat binding. In ascidians and mammals, ,-hexosaminidase released at fertilization from eggs has been proposed to modify sperm receptor glycoproteins of the egg envelope, thus setting up a block to polyspermy. Previously, it was shown that in potential sperm receptor glycoproteins of the ascidian Phallusia mammillata, N-acetylglucosamine is the prevailing glycoside residue and that the egg harbors three active molecular forms of ,-hexosaminidase. In the present study, P. mammillata,-hexosaminidase cDNA was isolated from an ovarian cDNA library and characterized. The deduced amino acid sequence showed a high similarity with other known ,-hexosaminidases; however, P. mammillata,-hexosaminidase had a unique potential N-glycosylation site. A phylogenetic analysis suggested that P. mammillata,-hexosaminidase developed independently after having branched off from the common ancestor gene of the chordate enzyme before two isoforms of the mammalian enzyme appeared. In situ hybridization revealed stage-specific expression of ,-hexosaminidase mRNA during oogenesis in the oocyte and in the accessory test and follicle cells. This suggests that the three egg ,-hexosaminidase forms are specific for the oocyte, test cells and follicle cells. [source] Molecular cloning of CYP1A gene and its expression by benzo(a)pyrene from goldfish (Carassius auratus)ENVIRONMENTAL TOXICOLOGY, Issue 3 2009Seung-Min Oh Abstract We cloned and sequenced the cytochrome P450 1A (CYP1A) gene from goldfish (Carassius auratus). It has a 1581 bp open reading frame that encodes a 526 amino acid protein with a theoretical molecular weight of 59.02 kDa. The CYP1A amino acid sequence clusters in a monophyletic group with other fish CYP1As, and more closely related to zebrafish CYP1A (91% identity) than to other fish CYP1As. Exposure to benzo(a)pyrene (BaP) by intraperitoneal injection increased biliary BaP metabolites and liver CYP1A gene expression. BaP exposure also increased CYP1A gene expression in extrahepatic organs, including intestine, and gill, which are sensitive to aqueous and dietary exposure to Arylhydrocarbon receptor (AhR) agonists. Therefore, goldfish CYP1A identified in this study offers basic information for further research related to biomarker use of CYP1A of goldfish. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2009. [source] Molecular cloning of cytochrome P4501A cDNA of medaka (Oryzias latipes) and messenger ribonucleic acid regulation by environmental pollutantsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 4 2004Jisung Ryu Abstract The sequence of cytochrome P4501A (CYP1A) cDNA of medaka (Oryzias latipes) was determined, and its messenger ribonucleic acid (mRNA) regulation by ,-naphthoflavone (,NF) was evaluated. The determined cDNA sequence contained 2,349 base pairs (bp), and the open reading frame contained a total of 1,563 bp encoding 521 predicted amino acids. The induction of CYP1A mRNA in medaka was evaluated using reverse transcription,polymerase chain reaction. The concentration,dependent induction of CYP1A mRNA in the liver was observed after exposure to ,NF at nominal concentrations of 20, 100, and 500 ,g/ L for 2 d. Time-dependent changes of CYP1A mRNA levels were also observed in the liver, gill, gut, and caudal fin tissues of medaka exposed to 100 ,g/L of ,NF for 7 d. Our results showed that the degree of CYP1A mRNA induction in the gill, gut, and caudal fin after exposure to ,NF was relatively higher than that in the liver, possibly because of low basal levels of CYP1A mRNA in the gill, gut, and caudal fin of nonexposed fish. The induction of medaka CYP1A mRNA was also observed after exposure to an environmental sample, landfill leachate. The CYP1A mRNA inductions in the gill, gut, and caudal fin were also higher than that in the liver as shown in the ,NF-treated groups. These results show that CYP1A mRNA determination in the gill, gut, and caudal fin, which are in direct contact with the polluted water, may become a useful method for monitoring CYP1A-inducible chemicals. [source] Molecular cloning and expression regulation of PRG-3, a new member of the plasticity-related gene familyEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2004Nicolai E. Savaskan Abstract Phospholipid-mediated signalling on neurons provokes diverse responses such as neurogenesis, pattern formation and neurite remodelling. We have recently uncovered a novel set of molecules in the mammalian brain, named plasticity-related genes (PRGs), which mediate lipid phosphate phosphatase activity and provide evidence for their involvement in mechanisms of neuronal plasticity. Here, we report on a new member of the vertebrate-specific PRG family, which we have named plasticity-related gene-3 (PRG-3). PRG-3 is heavily expressed in the brain and shows a specific expression pattern during brain development where PRG-3 expression is found predominantly in neuronal cell layers and is already expressed at embryonic day 16. In the mature brain, strongest PRG-3 expression occurs in the hippocampus and cerebellum. Overexcitation of neurons induced by kainic acid leads to a transient down-regulation of PRG-3. Furthermore, PRG-3 is expressed on neurite extensions and promotes neurite growth and a spreading-like cell body in neuronal cells and COS-7 cells. In contrast to previously described members of the PRG family, PRG-3 does not perform its function through enzymatic phospholipid degradation. In summary, our findings feature a new member of the PRG family which shows dynamic expression regulation during brain development and neuronal excitation. [source] Molecular cloning of the ecdysone receptor and the retinoid X receptor from the scorpion Liocheles australasiaeFEBS JOURNAL, Issue 23 2007Yoshiaki Nakagawa cDNAs of the ecdysone receptor and the retinoid X receptor were cloned from the Japanese scorpion Liocheles australasiae, and the amino acid sequences were deduced. The full-length cDNA sequences of the L. australasiae ecdysone receptor and the L. australasiae retinoid X receptor were 2881 and 1977 bp in length, respectively, and the open reading frames encoded proteins of 560 and 414 amino acids. The amino acid sequence of the L. australasiae ecdysone receptor was similar to that of the ecdysone receptor-A of the soft tick, Ornithodoros moubata (68%) and to that of the ecdysone receptor-A1 of the lone star tick, Amblyomma americanum (66%), but showed lower similarity to the ecdysone receptors of Orthoptera and Coleoptera (53,57%). The primary sequence of the ligand-binding region of the L. australasiae ecdysone receptor was highly homologous to that of ticks (85,86%). The amino acid sequence of the L. australasiae retinoid X receptor was also homologous to the amino acid sequence of ultraspiracles of ticks (63%) and insects belonging to the orders Orthoptera and Coleoptera (60,64%). The identity of both the L. australasiae ecdysone receptor and the L. australasiae retinoid X receptor to their lepidopteran and dipteran orthologs was less than 50%. The cDNAs of both the L. australasiae ecdysone receptor (L. australasiae ecdysone receptor-A) and the L. australasiae retinoid X receptor were successfully translated in vitro using a rabbit reticulocyte lysate system. An ecdysone analog, ponasterone A, bound to L. australasiae ecdysone receptor-A (KD = 4.2 nm), but not to L. australasiae retinoid X receptor. The L. australasiae retinoid X receptor did not enhance the binding of ponasterone A to L. australasiae ecdysone receptor-A, although L. australasiae retinoid X receptor was necessary for the binding of L. australasiae ecdysone receptor-A to ecdysone response elements. [source] Molecular cloning, expression and characterization of cDNA encoding cis -prenyltransferases from Hevea brasiliensisFEBS JOURNAL, Issue 23 2003A key factor participating in natural rubber biosynthesis Natural rubber from Hevea brasiliensis is a high molecular mass polymer of isoprene units with cis -configuration. The enzyme responsible for the cis -1,4-polymerization of isoprene units has been idengified as a particle-bound rubber transferase, but no gene encoding this enzyme has been cloned from rubber-producing plants. By using sequence information from the conserved regions of cis -prenyl chain elongating enzymes that were cloned recently, we have isolated and characterized cDNAs from H. brasiliensis for a functional factor participating in natural rubber biosynthesis. Sequence analysis revealed that all of the five highly conserved regions among cis -prenyl chain elongating enzymes were found in the protein sequences of the Hevea cis -prenyltransferase. Northern blot analysis indicated that the transcript(s) of the Hevea cis -prenyltransferase were expressed predominantly in the latex as compared with other Hevea tissues examined. In vitro rubber transferase assays using the recombinant gene product overexpressed in Escherichia coli revealed that the enzyme catalyzed the formation of long chain polyprenyl products with approximate sizes of 2 × 103,1 × 104 Da. Moreover, in the presence of washed bottom fraction particles from latex, the rubber transferase activity producing rubber product of high molecular size was increased. These results suggest that the Hevea cis -prenyltransferase might require certain activation factors in the washed bottom fraction particles for the production of high molecular mass rubber. [source] Molecular cloning and functional expression of a gene encoding an antiarrhythmia peptide derived from the scorpion toxinFEBS JOURNAL, Issue 18 2002Fang Peng From a cDNA library of Chinese scorpion Buthus martensii Karsch, full-length cDNAs of 351 nucleotides encoding precursors (named BmKIM) that contain signal peptides of 21 amino acid residues, a mature toxin of 61 residues with four disulfide bridges, and an extra Gly-Lys-Lys tail, were isolated. The genomic sequence of BmKIM was cloned and sequenced; it consisted of two exons disrupted by an intron of 1622 bp, the largest known in scorpion toxin genomes, inserted in the region encoding the signal peptide. The cDNA was expressed in Escherichia coli. The recombinant BmKIM was toxic to both mammal and insects. This is the first report that a toxin with such high sequence homology with an insect-specific depressant toxin group exhibits toxicity to mammals. Using whole cell patch-clamp recording, it was discovered that the recombinant BmKIM inhibited thesodium current in rat dorsal root ganglion neurons andventricular myocytes and protected against aconitine- induced cardiac arrhythmia. [source] A cocaine insensitive chimeric insect serotonin transporter reveals domains critical for cocaine interactionFEBS JOURNAL, Issue 16 2002Sumandeep K. Sandhu Serotonin transporters are key target sites for clinical drugs and psychostimulants, such as fluoxetine and cocaine. Molecular cloning of a serotonin transporter from the central nervous system of the insect Manduca sexta enabled us to define domains that affect antagonist action, particularly cocaine. This insect serotonin transporter transiently expressed in CV-1 monkey kidney cells exhibits saturable, high affinity Na+ and Cl, dependent serotonin uptake, with estimated Km and Vmax values of 436 ± 19 nm and 3.8 ± 0.6 × 10,18 mol·cell·min,1, respectively. The Manduca high affinity Na+/Cl, dependent transporter shares 53% and 74% amino acid identity with the human and fruit fly serotonin transporters, respectively. However, in contrast to serotonin transporters from these two latter species, the Manduca transporter is inhibited poorly by fluoxetine (IC50 = 1.23 µm) and cocaine (IC50 = 12.89 µm). To delineate domains and residues that could play a role in cocaine interaction, the human serotonin transporter was mutated to incorporate unique amino acid substitutions, detected in the Manduca homologue. We identified a domain in extracellular loop 2 (amino acids 148,152), which, when inserted into the human transporter, results in decreased cocaine sensitivity of the latter (IC50 = 1.54 µm). We also constructed a number of chimeras between the human and Manduca serotonin transporters (hSERT and MasSERT, respectively). The chimera, hSERT1,146/MasSERT106,587, which involved N-terminal swaps including transmembrane domains (TMDs) 1 and 2, was remarkably insensitive to cocaine (IC50 = 180 µm) compared to the human (IC50 = 0.431 µm) and Manduca serotonin transporters. The chimera MasSERT1,67/hSERT109,630, which involved only the TMD1 swap, showed greater sensitivity to cocaine (IC50 = 0.225 µm) than the human transporter. Both chimeras showed twofold higher serotonin transport affinity compared to human and Manduca serotonin transporters. Our results show TMD1 and TMD2 affect the apparent substrate transport and antagonist sensitivity by possibly providing unique conformations to the transporter. The availability of these chimeras facilitates elucidation of specific amino acids involved in interactions with cocaine. [source] Molecular cloning of the Matrix Gla Protein gene from Xenopus laevisFEBS JOURNAL, Issue 7 2002Functional analysis of the promoter identifies a calcium sensitive region required for basal activity To analyze the regulation of Matrix Gla Protein (MGP) gene expression in Xenopus laevis, we cloned the xMGP gene and its 5, region, determined their molecular organization, and characterized the transcriptional properties of the core promoter. The Xenopus MGP (xMGP) gene is organized into five exons, one more as its mammalian counterparts. The first two exons in the Xenopus gene encode the DNA sequence that corresponds to the first exon in mammals whereas the last three exons show homologous organization in the Xenopus MGP gene and in the mammalian orthologs. We characterized the transcriptional regulation of the xMGP gene in transient transfections using Xenopus A6 cells. In our assay system the identified promoter was shown to be transcriptionally active, resulting in a 12-fold induction of reporter gene expression. Deletional analysis of the 5, end of the xMGP promoter revealed a minimal activating element in the sequence from ,70 to ,36 bp. Synthetic reporter constructs containing three copies of the defined regulatory element delivered 400-fold superactivation, demonstrating its potential for the recruitment of transcriptional activators. In gel mobility shift assays we demonstrate binding of X. laevis nuclear factors to an extended regulatory element from ,180 to ,36, the specificity of the interaction was proven in competition experiments using different fragments of the xMGP promoter. By this approach the major site of factor binding was demonstrated to be included in the minimal activating promoter fragment from ,70 to ,36 bp. In addition, in transient transfection experiments we could show that this element mediates calcium dependent transcription and increasing concentrations of extracellular calcium lead to a significant dose dependent activation of reporter gene expression. [source] Molecular cloning and heterologous expression of novel glucosyltransferases from tobacco cultured cells that have broad substrate specificity and are induced by salicylic acid and auxinFEBS JOURNAL, Issue 14 2001Goro Taguchi Scopoletin is one of the phytoalexins in tobacco. Cells of the T-13 cell line (Nicotiana tabacum L. Bright Yellow) accumulate a large amount of scopoletin, also known as 7-hydroxy-6-methoxycoumarin, as a glucoconjugate, scopolin, in vacuoles. We report here the molecular cloning of glucosyltransferases that can catalyze the glucosylation of many kinds of secondary metabolites including scopoletin. Two cDNAs encoding glucosyltransferase (NtGT1a and NtGT1b) were isolated from a cDNA library derived from the tobacco T-13 cell line by screening with heterologous cDNAs as a probe. The deduced amino-acid sequences of NtGT1a and NtGT1b exhibited 92% identity with each other, ,,20,50% identities with other reported glucosyltransferases. Heterologous expression of these genes in Escherichia coli showed that the recombinant enzymes had glucosylation activity against both flavonoids and coumarins. They also strongly reacted with 2-naphthol as a substrate. These recombinant enzymes can utilize UDP-glucose as the sugar donor, but they can also utilize UDP-xylose as a weak donor. RNA blot analysis showed that these genes are induced by salicylic acid and auxin, but the time course of the expression was different. This result is similar to the changes in scopoletin glucosylation activity in these tobacco cells after addition of these plant growth regulators. These results might suggest that one of the roles of the products of these genes is scopoletin glucosylation, in response to salicylic acid and/or auxin, together with the other glucosyltransferases in tobacco cells. [source] Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastorisFEBS JOURNAL, Issue 6 2000Ludovic Otterbein Laccases are multicopper-containing enzymes which catalyse the oxidation of phenolic and nonphenolic compounds with the concomitant reduction of molecular oxygen. In this study, a full-length cDNA coding for laccase (lac1) from Pycnoporus cinnabarinus I-937 was isolated and characterized. The corresponding open reading frame is 1557 nucleotides long and encodes a protein of 518 amino acids. The cDNA encodes a precursor protein containing a 21 amino-acid signal sequence corresponding to a putative signal peptide. The deduced amino-acid sequence of the encoded protein was similar to that of other laccase proteins, with the residues involved in copper coordination sharing the greatest extent of similarity. The cDNA encoding for laccase was placed under the control of the alcohol oxidase (Aox 1) promoter and expressed in the methylotropic yeast Pichia pastoris. The laccase leader peptide, as well as the Saccharomyces cerevisiae,-factor signal peptide, efficiently directed the secretion into the culture medium of laccase in an active form. Moreover, the laccase activity was directly detected in plates. The identity of the recombinant product was further confirmed by protein immunoblotting. The expected molecular mass of the mature protein is 81 kDa. However, the apparent molecular mass of the recombinant protein is 110 k Da, thus suggesting that the protein expressed in P. pastoris may be hyperglycosylated. [source] Retinoid signaling and cardiac anteroposterior segmentationGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 3 2001José Xavier-Neto Abstract Summary: Establishment of anterior,posterior polarity is one of the earliest decisions in cardiogenesis. Specification of anterior (outflow) and posterior (inflow) structures ensures proper connections between venous system and inflow tract and between arterial tree and outflow tract. The last few years have witnessed remarkable progress in our understanding of cardiac anteroposterior patterning. Molecular cloning and subsequent studies on RALDH2, the key embryonic retinaldehyde dehydrogenase in retinoic acid (RA) synthesis, provided the missing link between teratogenic studies on RA deficiency and excess and normal chamber morphogenesis. We discuss work establishing the foundations of our current understanding of the mechanisms of cardiac anteroposterior segmentation, the reasons why early evidence pointing to the role of RA in anteroposterior segmentation was overlooked, and the key experiments unraveling the role of RA in cardiac anteroposterior segmentation. We have also integrated recent experiments in a model of cardiac anteroposterior patterning in which RALDH2 expression determines anteroposterior boundaries in the heart field. genesis 31:97,104, 2001. © 2001 Wiley-Liss, Inc. [source] Molecular cloning, characterization, expression pattern and cellular distribution of an ovarian lipophorin receptor in the cockroach, Leucophaea maderaeINSECT MOLECULAR BIOLOGY, Issue 3 2009M. Tufail Abstract A cDNA that encodes a lipophorin receptor (LpR) with a predicted structure similar to that of the low density lipoprotein receptor (LDLR) gene superfamily was cloned from ovaries of the cockroach, Leucophaea maderae (Lem) and characterized. This is the first LpR sequenced from the order Dictyoptera. The cDNA has a length of 3362 bp coding for an 888-residue mature protein with a predicted molecular mass of ~99.14 kDa and a pI value of 4.68. The deduced amino acid sequence showed that the LemLpR harbours eight ligand-binding repeats (LBRs) at the N-terminus similar to the other insect LpRs, and thus resembles vertebrate VLDLRs. In addition to eight tandemly arranged LBRs, the five-domain receptor contains an O -linked sugar region and the classic LDLR internalization signal, FDNPVY. Northern blot analysis revealed the presence of ~4.0 kb ovarian mRNA that was transcribed throughout oogenesis with its peak especially during late previtellogenic and vitellogenic periods (from days 3 to 11). LpR transcript(s) or homologues of LDLRs were also detected in the head, midgut, Malpighian tubules, muscles and in the fat body. RNA in situ hybridization and immunocytochemistry localized the LpR mRNA and protein to germ line-derived cells, the oocytes, and revealed that LpR gene transcription and translation starts very early during oocyte differentiation in the germarium. LpR protein was evenly distributed throughout the cytoplasm during previtellogenic periods of oogenesis. However, during vitellogenic stages, the receptor was accumulated mainly in the cortex of the oocyte. Immunoblot analysis probed an ovarian LpR protein of ~115 and 97 kDa under reducing and nonreducing conditions, respectively. The protein signal appeared on day 2, increased every day and was high during vitellogenic periods from day 4 to day 7. Southern blot analysis suggested the presence of a single copy of the LpR gene in the genome of Le. maderae. [source] Molecular cloning and immunolocalization of a diuretic hormone receptor in rice brown planthopper (Nilaparvata lugens)INSECT MOLECULAR BIOLOGY, Issue 5 2004D. R. G. Price Abstract RNA extracted from guts of rice brown planthopper, Nilaparvata lugens, was used to clone cDNA predicted to encode a diuretic hormone receptor (DHR). The DHR, a member of the calcitonin/secretin/corticotropin-releasing factor family of G-protein-coupled receptors, contains seven transmembrane domains and a large N-terminal extracellular domain potentially involved in hormone binding. The N-terminal domain was expressed as a recombinant protein, purified and used to raise antibodies. Anti-DHR IgG bound specifically to Malpighian tubules in immunolocalization experiments using dissected guts, and to a putative DHR polypeptide from N. lugens gut on Western blots. Anti-DHR IgG delivered orally to insects was not detected in the haemolymph, and showed no binding to gut or tubules, confirming that DHR N-terminal hormone-binding domain is not exposed to the gut lumen. [source] Molecular cloning of two prophenoloxidase genes from the mosquito Aedes aegyptiINSECT MOLECULAR BIOLOGY, Issue 1 2001A. S. Taft Abstract The biosynthesis of melanotic materials is an important process in the life of a mosquito. Melanin production is critical for many diverse processes such as egg chorion tanning, cuticular sclerotization, and melanotic encapsulation of metazoan parasites. Prophenoloxidase plays a critical role in this biochemical cascade. Two cDNAs, one full length and one partial clone, and two genomic clones encoding prophenoloxidase (pro-PO) were isolated from the yellow fever mosquito, Aedes aegypti. The full-length cDNA, pAaProPO1, is 2286 bp long with a 2055 bp open reading frame encoding a 685 amino acid protein that shares 89% identity with Armigeres subalbatus pro - PO. It contains two putative copper binding domains (amino acids 197,243 and 346,423) that are homologous to other insect pro-POs. AaProPO1 messenger RNA (mRNA) was detected by reverse transcription polymerase chain reaction (RT-PCR) only from third-stage larvae and not in adult mosquitoes after blood feeding, during the melanotic encapsulation of Dirofilaria immitis microfilariae or following exposure to bacteria. A 750 bp fragment of the second cDNA (pAaProPO2) was cloned using RT-PCR from mRNA obtained from 14-day postovipostional eggs. AaProPO2 mRNA was not found in any other life stages, and may be in low abundance or transiently expressed. AaProPO2 and AaProPO1 each contain three introns that are 60, 68 and 58 bp and 61, 69 and 59 bp long, respectively, and the intron sequences of these two genes are not similar. [source] Molecular cloning and expression of Tenebrio molitor ultraspiracle during metamorphosis and in vivo induction of its phosphorylation by 20-hydroxyecdysoneINSECT MOLECULAR BIOLOGY, Issue 3 2000M. Nicolaď Abstract Using a RT-PCR approach, the Tenebrio molitor homologue of Drosophila Ultraspiracle (TmUSP) was characterized. Its DNA binding domain shows a degree of identity with those of the other insect USPs. However, the ligand binding domain is closer to those of retinoid X receptors. Using an antibody raised against DmUSP, Western blot analysis of proteins from epidermis and other tissues revealed five immunoreactive bands, corresponding to different phosphorylated forms of a unique polypeptide, as shown by ,-phosphatase treatment. The nuclear form of TmUSP seems unphosphorylated. An in vivo 20-hydroxyecdysone treatment increases considerably and rapidly the phosphorylated forms of TmUSP. This post-translational modification may play a role in the 20-hydroxyecdysone response. [source] Molecular cloning of several rat ABC transporters including a new ABC transporter, Abcb8, and their expression in rat testisINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 3 2006Nathalie Melaine Summary Several members of the ABC transporter superfamily play an important role in testicular physiology and defence against anticancer drugs. Using a reverse transcription-polymerase chain reaction strategy with degenerate primers and rat testis RNA as template, we have looked for the presence of other members of this superfamily. Of the six partial cDNA found, five corresponded to ABC transporters already known ,Mdr1b, Mrp1, Tapl/Abcb9, Umat/Abcb6 and Sur2/Abcc9, and one presented a strong homology with mouse and human ABCB8. Using a 5, and 3, RACE approach, we cloned the full-length cDNA and found that the predicted protein presented 92% and 80% homology with the mouse and human proteins respectively. Strong expression of rat abcb8 was found in heart, brain and testis when compared with liver, lung and spleen. In the testis, rat abcb8 was expressed both in the somatic Sertoli cells and peritubular cells and in the germline (spermatogonia and pachytene spermatocytes). Furthermore, Umat/Abcb6 was very highly expressed in the testis (high amounts in meiotic pachytene spermatocytes and low amount in post-meiotic early spermatids). In conclusion, we confirm the presence of several ABC transporters in the testis and also provide evidence of the presence of Abcb8 in the organ. [source] Molecular cloning of feline tumour necrosis factor receptor type I (TNFR I) and expression of TNFR I and TNFR II in lymphoid cells in catsINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 2 2003T. Mizuno Summary Tumour necrosis factor (TNF)-, is a pro-inflammatory cytokine produced by many types of cells. It has been shown that two distinct TNF receptors (TNFRs), TNFR type I (TNFR I) and TNFR type II (TNFR II), have different functions in signal transduction, which is possibly associated with the development of a variety of diseases. In this study, we isolated a feline TNFR I cDNA clone and analysed the expression of TNFR I and TNFR II mRNA in feline lymphoid cells. The deduced amino acid sequence of feline TNFRI cDNA showed 75.8, 62.5 60.9 and 72.1% similarity with those of its human, mouse, rat, and pig counterparts, respectively. The feline TNFR I cDNA was shown to encode extracellular, transmembrane and intracellular domains fundamentally conserved in the homologues of other species. Expression of TNFR I and TNFR II mRNAs was shown to be up-regulated in feline peripheral blood mononuclear cells (PBMC) by stimulation with concanavalin A. Five of six feline lymphoma cell lines were shown to express both TNFR I and TNFR II mRNAs. The expression of TNFR I in PBMC was up-regulated in cats infected with feline immunodeficiency virus (FIV), whereas the expression of TNFR II in PBMC was not different between FIV-infected cats and uninfected cats. The present study indicate that expression of TNFR I and TNFR II may be associated with disease progression, especially in retrovirus infections in cats. [source] Molecular cloning of four lambda light chain cDNAs from the Australian brushtail possum Trichosurus vulpeculaINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 2 2002K. Belov Summary A brushtail possum mesenteric lymph node cDNA library was screened with a grey short-tail opossum C, probe and four immunoglobulin lambda cDNAs were isolated. Two of the isolated clones (L5 and L10) contained identical framework 4 regions and constant regions (but different variable regions), suggesting that the possum lambda locus is organized as multiple J-C pairs , a feature seen in the opossum and placental mammals. The cloning of the lambda light chain cDNAs signifies the completion of the basic molecular characterization of the brushtail possum immunoglobulin repertoire. The availability of this sequence data will allow extensive analysis of the immune response of the brushtail possum at the molecular level, as well as the development of specific immunological reagents for detection of immunoglobulin molecules at the protein level. [source] Molecular cloning of the cDNAs encoding the feline B-lymphocyte activation antigen B7,1 (CD80) and B7,2 (CD86) homologues which interact with human CTLA4-IgINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 5-6 2000Y. Nishimura We cloned the cDNAs encoding the feline homologues of B-lymphocyte activation antigens B7,1 (CD80) and B7,2 (CD86). We expressed recombinant feline CD80 and CD86 molecules by the baculovirus expression system, and demonstrated their binding ability to human CTLA4-murine immunoglobulin fusion protein. [source] Molecular cloning and characterization of bovine PRKAG3 gene: structure, expression and single nucleotide polymorphism detectionJOURNAL OF ANIMAL BREEDING AND GENETICS, Issue 5 2005S.L. Yu Summary The protein kinase adenosine monophosphate-activated ,3-subunit (PRKAG3) gene encodes a muscle-specific isoform of the regulatory gamma-subunit of adenosine monophosphate-activated protein kinase, which plays a key role in regulating energy homeostasis in eucaryotes. It is well known that mutations in the PRKAG3 gene affect high glycogen content in the porcine skeletal muscle and, consequently, meat quality. The genomic structure and sequence of the bovine PRKAG3 were analysed from a Korean cattle BAC clone. The bovine PRKAG3 gene comprises 13 exons and spans approximately 6.8 kb on BTA2. From 5, and 3,-rapid amplification of cDNA ends experiments, the full-length cDNA of bovine PRKAG3 has been identified, encoding a deduced protein of 465 amino acids. Two splice isoforms, generated by the alternative splicing of exon 2, were also identified. Northern blot analysis demonstrated that, similar to other species, the bovine PRKAG3 transcript was only expressed in skeletal muscle. Seven single nucleotide polymorphisms, including two previously identified variants, were detected in four Bos taurus cattle breeds. The bovine PRKAG3 gene described in this study may be involved in muscle-related genetic diseases or meat quality traits in cattle. [source] Molecular cloning and characterization of alpha-class glutathione S -transferase gene from the liver of silver carp, bighead carp, and other major chinese freshwater fishesJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 3 2006Wan-Qin Liao Abstract Two full-length cDNAs encoding glutathione S -transferase (GST) were cloned and sequenced from the hepatopancreas of planktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis). The silver carp and bighead carp GST cDNA were 920 and 978 bp in length, respectively, and both contained an open reading frame that encoding 223 amino acids. Partial GST cDNA sequences were also obtained from the liver of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius auratu), mud carp (Cirrhinus molitorella), and tilapia (Oreochromis nilotica). All these GSTs could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. The three-dimensional structure of the silver carp GST was predicted using a computer program, and was found to fit the classical two-domain GST structure. Using the genome walker method, a 875-bp 5,-flanking region of the silver carp GST gene was obtained, and several lipopolysaccharide (LPS) response elements were identified in the promoter region of the phytoplanktivorous fish GST gene, indicating that the GST gene expression of this fish might be regulated by LPS, released from the toxic blue-green algae producing microcystins. To compare the constitutive expression level of the liver GST gene among the six freshwater fishes with completely different tolerance to microcystins, beta-actin was used as control and the ratio GST/beta-actin mRNA (%) was determined as 130.7 ± 6.6 (grass carp), 103.1 ± 8.9 (bighead carp), 92.6 ± 15.0 (crucian carp), 72.3 ± 7.8 (mud carp), 58.8 ± 11.5 (silver carp), and 33.6 ± 13.7 (tilapia). The constitutive expression level of the liver GST gene clearly shows that all the six freshwater fishes had a negative relationship with their tolerance to microcystins: high-resistant fishes (phytoplanktivorous silver carp and tilapia) had the lowest tolerance to microcystins and the high-sensitive fish (herbivorous grass carp) had the highest tolerance to microcystins. Taken together with the reciprocal relationship of constitutive and inducible liver GST expression level in some of the tested fish species to microcystin exposure, a molecular mechanism for different microcystin detoxification abilities of the warm freshwater fishes was discussed. © 2006 Wiley Periodicals, Inc. J Biochem Mol Toxicol 20:114,126, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20125 [source] Molecular cloning, genomic organization and functional characterization of a new short-chain potassium channel toxin-like peptide BmTxKS4 from Buthus martensii Karsch(BmK)JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 4 2004Sheng Jiqun Abstract Scorpion venom contains many small polypeptide toxins, which can modulate Na+, K+, Cl,, and Ca2+ ion,channel conductance in the cell membrane. A full-length cDNA sequence encoding a novel type of K+ -channel toxin (named BmTxKS4) was first isolated and identified from a venom gland cDNA library of Buthus martensii Karsch (BmK). The encoded precursor contains 78 amino acid residues including a putative signal peptide of 21 residues, propeptide of 11 residues, and a mature peptide of 43 residues with three disulfide bridges. BmTxKS4 shares the identical organization of disulfide bridges with all the other short-chain K+ -channel scorpion toxins. By PCR amplification of the genomic region encoding BmTxKS4, it was shown that BmTxKS4 composed of two exons is disrupted by an intron of 87 bp inserted between the first and the second codes of Phe (F) in the encoding signal peptide region, which is completely identical with that of the characterized scorpion K+ -channel ligands in the size, position, consensus junctions, putative branch point, and A+T content. The GST-BmTxKS4 fusion protein was successfully expressed in BL21 (DE3) and purified with affinity chromatography. About 2.5 mg purified recombinant BmTxKS4 (rBmTxKS4) protein was obtained by treating GST-BmTxKS4 with enterokinase and sephadex chromatography from 1 L bacterial culture. The electrophysiological activity of 1.0,M rBmTxKS4 was measured and compared by whole cell patch-clamp technique. The results indicated that rBmTxKS4 reversibly inhibited the transient outward K+ current (Ito), delayed inward rectifier K+ current (Ik1), and prolonged the action potential duration of ventricular myocyte, but it has no effect on the action potential amplitude. Taken together, BmTxKS4 is a novel subfamily member of short-strain K+ -channel scorpion toxin. © 2004 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:187,195, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20026 [source] Molecular cloning and functional analysis of Photobacterium damselae subsp. piscicida haem receptor geneJOURNAL OF FISH DISEASES, Issue 2 2005H Naka Abstract A haem receptor gene from Photobacterium damselae subsp. piscicida (formerly known as Pasteurella piscicida) has been cloned, sequenced and analysed for its function. The gene, designated as pph, has an open reading frame consisting of 2154 bp, a predicted 718 amino acid residues and exists as a single copy. It is homologous with the haem receptors of Vibrio anguillarum hupA, V. cholerae hutA, V. mimicus mhuA and V. vulnificus hupA at 32.7, 32.7, 45.6 and 30.9%, respectively, and is highly conserved, consisting of a Phe-Arg-Ala-Pro sequence (FRAP), an iron transport related molecule (TonB) and a Asn-Pron-Asn-Leu sequence (NPNL), binding motifs associated with haem receptors. As a single gene knockout mutant P. damselae subsp. piscicida was able to bind haem in the absence of pph, suggesting that other receptors may be involved in its iron transport system. This study shows that the P. damselae subsp. piscicida pph belongs to the haem receptor family, is conserved and that its iron-binding system may involve more than one receptor. [source] Molecular cloning of Yersinia ruckeri aroA gene: a useful taxonomic toolJOURNAL OF FISH DISEASES, Issue 7 2001J Yugueros The aroA gene of Yersinia ruckeri, which encodes 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase was cloned by complementation of the aroA mutation in Escherichia coli AB2829 by using pUC18 plasmid as a vector. Nucleotide sequence of the aroA gene revealed an open reading frame of 427 amino acids showing a high degree of homology to other bacterial AroA proteins. A pair of primers with 23 and 20 nucleotides were selected from the 5, and 3, termini, respectively, and formed the basis of a specific polymerase chain reaction (PCR) assay. A 1165-bp deoxyribonucleic acid (DNA) fragment was amplified from all lysed Y. ruckeri strains. An identical size fragment was also amplified from lysed Y. pseudotuberculosis, Y. aldovae, Salmonella enteritidis and E. coli, but not from other enterobacteria. AluI restriction fragment length polymorphism (RFLP) of the PCR amplified products allowed for differentiation between Y. ruckeri and the other bacteria. Specificity and sensitivity make this PCR assay a useful method for rapid identification and diagnosis of Y. ruckeri infections. [source] Molecular cloning of three nonhuman primate follicle stimulating hormone ,-subunit cDNAsJOURNAL OF MEDICAL PRIMATOLOGY, Issue 6 2001M.J. Wolfgang The follicle stimulating hormone (FSH) ,-subunit cDNAs were cloned and sequenced for an old world primate, the rhesus monkey (Macaca mulatta), and two New World primates, the common marmoset (Callithrix jacchus) and pygmy marmoset (Cebuella pygmaea). The cDNA and predicted amino acid sequences of the rhesus monkey FSH ,-subunit were related most closely to the human FSH , -subunit (>96% identity). The common and pygmy marmosets have identical FSH , -subunit cDNAs, whereas the marmoset FSH , -subunit diverges from the rhesus and human molecules with less than 93% identity. These results have significance for the implementation of assisted reproductive technologies in the nonhuman primate as well as the evolution of genes encoding reproductive hormones. [source] Functional implications for Kir4.1 channels in glial biology: from K+ buffering to cell differentiationJOURNAL OF NEUROCHEMISTRY, Issue 3 2008Michelle L. Olsen Abstract Astrocytes and oligodendrocytes are characterized by a very negative resting potential and a high resting permeability for K+ ions. Early pharmacological and biophysical studies suggested that the resting potential is established by the activity of inwardly rectifying, Ba2+ sensitive, weakly rectifying Kir channels. Molecular cloning has identified 16 Kir channels genes of which several mRNA transcripts and protein products have been identified in glial cells. However, genetic deletion and siRNA knock-down studies suggest that the resting conductance of astrocytes and oligodendrocytes is largely due to Kir4.1. Loss of Kir4.1 causes membrane depolarization, and a break-down of K+ and glutamate homeostasis which results in seizures and wide-spread white matter pathology. Kir channels have also been shown to act as critical regulators of cell division whereby Kir function is correlated with an exit from the cell cycle. Conversely, loss of functional Kir channels is associated with re-entry of cells into the cell cycle and gliosis. A loss of functional Kir channels has been shown in a number of neurological diseases including temporal lobe epilepsy, amyotrophic lateral sclerosis, retinal degeneration and malignant gliomas. In the latter, expression of Kir4.1 is sufficient to arrest the aberrant growth of these glial derived tumor cells. Kir4.1 therefore represents a potential therapeutic target in a wide variety of neurological conditions. [source] | |||||||||||||||||||||||||||||||