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cDNA Cloning (cdna + cloning)
Selected AbstractsPurification and cDNA Cloning of Lysozyme II from Cabbage Butterfly, Artogeia rapae LarvaeENTOMOLOGICAL RESEARCH, Issue 4 2005BANG In Seok ABSTRACT Last instar larvae of cabbage butterfly Artogeia rapae respond to injection of bacteria with a set of inducible antibacterial peptides/proteins. The inducible peptides/proteins are related to the known hinnavins (I and II) and lysozymes (I and II). The lysozyme II has been isolated by heat treatment, cation exchange, and reversed-phase chromatography from immunized hemolymph of last instar larvae. The lysozyme II gene of A. rapae was isolated and its nucleotide sequence was determined by the RACE-PCR from immunized fat body with E. coli. It has an open reading frame of 414 bp nucleotide corresponding to 138 amino acids including an 18 amino acid signal sequence. The molecular weight and the isoelectric point of Artogeia lysozyme II without a signal peptide were 13,649.38 Da and 9.11, respectively. It is great similarity with Manduca lysozyme among other lepidopteran. [source] Rnf19a, a ubiquitin protein ligase, and Psmc3, a component of the 26S proteasome, Tether to the acrosome membranes and the head,tail coupling apparatus during rat spermatid developmentDEVELOPMENTAL DYNAMICS, Issue 7 2009Eugene Rivkin Abstract We report the cDNA cloning of rat testis Rnf19a, a ubiquitin protein ligase, and show 98% and 93% protein sequence identity of testicular mouse and human Rnf19a, respectively. Rnf19a interacts with Psmc3, a protein component of the 19S regulatory cap of the 26S proteasome. During spermatid development, Rnf19a and Psmc3 are initially found in Golgi-derived proacrosomal vesicles. Later on, Rnf19a, Psmc3, and ubiquitin are seen along the cytosolic side of the acrosomal membranes and the acroplaxome, a cytoskeletal plate linking the acrosome to the spermatid nuclear envelope. Rnf19a and Psmc3 accumulate at the acroplaxome marginal ring,manchette perinuclear ring region during spermatid head shaping and in the developing sperm head,tail coupling apparatus and tail. Rnf19a and Psmc3 may interact directly or indirectly with each other, presumably pointing to the participation of the ubiquitin,proteasome system in acrosome biogenesis, spermatid head shaping, and development of the head-tail coupling apparatus and tail. Developmental Dynamics 238:1851,1861, 2009. © 2009 Wiley-Liss, Inc. [source] Characterization of a hemocyte intracellular fatty acid-binding protein from crayfish (Pacifastacus leniusculus) and shrimp (Penaeus monodon)FEBS JOURNAL, Issue 13 2006Irene Söderhäll Intracellular fatty acid-binding proteins (FABPs) are small members of the superfamily of lipid-binding proteins, which occur in invertebrates and vertebrates. Included in this superfamily are the cellular retinoic acid-binding proteins and retinol-binding proteins, which seem to be restricted to vertebrates. Here, we report the cDNA cloning and characterization of two FABPs from hemocytes of the freshwater crayfish Pacifastacus leniusculus and the shrimp Penaeus monodon. In both these proteins, the binding triad residues involved in interaction with ligand carboxylate groups are present. From the sequence and homology modeling, the proteins are probably FABPs and not retinoic acid-binding proteins. The crayfish transcript (plFABP) was detected at high level in hemocytes, hepatopancreas, intestine and ovary and at low level in hematopoietic tissue and testis. Its expression in hematopoietic cells varied depending on the state of the crayfish from which it was isolated. Expression was 10,15 times higher in cultures isolated from crayfish with red colored plasma, in which hemocyte synthesis was high, if retinoic acid was added to the culture medium. In normal colored crayfish, with normal levels of hemocytes, no increase in expression of p1FABP was detected. Two other putative plFABP ligands, stearic acid and oleic acid, did not have any effect on plFABP expression in hematopoietic cells. These results suggest that retinoic acid-dependent signaling may be present in crustaceans. [source] cDNA cloning and characterization of a novel calmodulin-like protein from pearl oyster Pinctada fucataFEBS JOURNAL, Issue 19 2005Shuo Li Calcium metabolism in oysters is a very complicated and highly controlled physiological and biochemical process. However, the regulation of calcium metabolism in oyster is poorly understood. Our previous study showed that calmodulin (CaM) seemed to play a regulatory role in the process of oyster calcium metabolism. In this study, a full-length cDNA encoding a novel calmodulin-like protein (CaLP) with a long C-terminal sequence was identified from pearl oyster Pinctada fucata, expressed in Escherichia coli and characterized in vitro. The oyster CaLP mRNA was expressed in all tissues tested, with the highest levels in the mantle that is a key organ involved in calcium secretion. In situ hybridization analysis reveals that CaLP mRNA is expressed strongly in the outer and inner epithelial cells of the inner fold, the outer epithelial cells of the middle fold, and the dorsal region of the mantle. The oyster CaLP protein, with four putative Ca2+ -binding domains, is highly heat-stable and has a potentially high affinity for calcium. CaLP also displays typical Ca2+ -dependent electrophoretic shift, Ca2+ -binding activity and significant Ca2+ -induced conformational changes. Ca2+ -dependent affinity chromatography analysis demonstrated that oyster CaLP was able to interact with some different target proteins from those of oyster CaM in the mantle and the gill. In summary, our results have demonstrated that the oyster CaLP is a novel member of the CaM superfamily, and suggest that the oyster CaLP protein might play a different role from CaM in the regulation of oyster calcium metabolism. [source] The N-glycans of yellow jacket venom hyaluronidases and the protein sequence of its major isoform in Vespula vulgarisFEBS JOURNAL, Issue 20 2005Daniel Kolarich Hyaluronidase (E.C. 3.2.1.35), one of the three major allergens of yellow jacket venom, is a glycoprotein of 45 kDa that is largely responsible for the cross-reactivity of wasp and bee venoms with sera of allergic patients. The asparagine-linked carbohydrate often appears to constitute the common IgE-binding determinant. Using a combination of MALDI MS and HPLC of 2-aminopyridine-labelled glycans, we found core-difucosylated paucimannosidic glycans to be the major species in the 43,45 kDa band of Vespula vulgaris and also in the corresponding bands of venoms from five other wasp species (V. germanica, V. maculifrons, V. pensylvanica, V. flavopilosa and V. squamosa). Concomitant peptide mapping of the V. vulgaris 43 kDa band identified the known hyaluronidase, Ves v 2 (SwissProt P49370), but only as a minor component. De novo sequencing by tandem MS revealed the predominating peptides to resemble a different, yet homologous, sequence. cDNA cloning retrieved a sequence with 58 and 59% homology to the previously known isoform and to the Dolichovespula maculata and Polistes annularis hyaluronidases. Close homologues of this new, putative hyaluronidase b (Ves v 2b) were also the major isoform in the other wasp venoms. [source] Purification, characterization, cDNA cloning and nucleotide sequencing of a cellulase from the yellow-spotted longicorn beetle, Psacothea hilarisFEBS JOURNAL, Issue 16 2003Masahiro Sugimura A cellulase (endo-,-1,4-glucanase, EC 3.2.1.4) was purified from the gut of larvae of the yellow-spotted longicorn beetle Psacothea hilaris by acetone precipitation and elution from gels after native PAGE and SDS/PAGE with activity staining. The purified protein formed a single band, and the molecular mass was estimated to be 47 kDa. The purified cellulase degraded carboxymethylcellulose (CMC), insoluble cello-oligosaccharide (average degree of polymerization 34) and soluble cello-oligosaccharides longer than cellotriose, but not crystalline cellulose or cellobiose. The specific activity of the cellulase against CMC was 150 µmol·min,1·(mg protein),1. TLC analysis showed that the cellulase produces cellotriose and cellobiose from insoluble cello-oligosaccharides. However, a glucose assay linked with glucose oxidase detected a small amount of glucose, with a productivity of 0.072 µmol·min,1·(mg protein),1. The optimal pH of P. hilaris cellulase was 5.5, close to the pH in the midgut of P. hilaris larvae. The N-terminal amino-acid sequence of the purified P. hilaris cellulase was determined and a degenerate primer designed, which enabled a 975-bp cDNA clone containing a typical polyadenylation signal to be obtained by PCR and sequencing. The deduced amino-acid sequence of P. hilaris cellulase showed high homology to members of glycosyl hydrolase family 5 subfamily 2, and, in addition, a signature sequence for family 5 was found. Thus, this is the first report of a family 5 cellulase from arthropods. [source] Regulated expression and intracellular localization of cystatin F in human U937 cellsFEBS JOURNAL, Issue 22 2002Carl-Michael Nathanson Cystatin F is a cysteine peptidase inhibitor recently discovered in haematopoietic cells by cDNA cloning. To further investigate the expression, distribution and properties of the native human inhibitor the promyeloid cell line U937 has been studied. The cells expressed relatively large quantities of cystatin F, which was found both secreted and intracellularly. The intracellular levels were unusually high for a secreted cystatin (, 25% of the cystatin F in 2- or 4-day culture medium). By contrast, U937 cells contained only 3,4% of the related inhibitor, cystatin C. Cystatin F purified from lysates of U937 cells showed three major forms carrying two, one or no carbohydrate chains. Immunocytochemistry demonstrated a marked cytoplasmic cystatin F staining in a granular pattern. Double staining with a marker for endoplasmic reticulum revealed no colocalization for cystatin F. Analysis of the promoter region of the cystatin F gene (CST7) showed that it, like that of the cystatin C gene (CST3), is devoid of typical TATA- and CAAT-box elements. In contrast to the cystatin C promoter, it does not contain multiple Sp1 binding sites, but has a unique site for C/EBP,, possibly explaining the restricted expression of the cystatin F gene. Cells stimulated with all- trans retinoic acid to differentiate them towards a granulocytic pathway, showed a strong (, 18-fold) down-regulation of intracellular cystatin F and almost abolished secreted levels of the inhibitor. Stimulation with tetradecanoyl phorbol acetate, causing monocytic differentiation, also resulted in down-regulation (two fold to threefold) of cystatin F expression, whereas the cystatin C expression was essentially unaltered in both experiments. The results suggest that cystatin F as an intracellular cysteine peptidase inhibitor with readily regulated expression, may be a candidate to control the cysteine peptidase activity known to be essential for antigen presentation in different blood cell lineages. [source] Cloning and characterization of novel snake venom proteins that block smooth muscle contractionFEBS JOURNAL, Issue 11 2002Yasuo Yamazaki In this study, we isolated a 25-kDa novel snake venom protein, designated ablomin, from the venom of the Japanese Mamushi snake (Agkistrodon blomhoffi). The amino-acid sequence of this protein was determined by peptide sequencing and cDNA cloning. The deduced sequence showed high similarity to helothermine from the Mexican beaded lizard (Heloderma horridum horridum), which blocks voltage-gated calcium and potassium channels, and ryanodine receptors. Ablomin blocked contraction of rat tail arterial smooth muscle elicited by high K+ -induced depolarization in the 0.1,1 µm range, but did not block caffeine-stimulated contraction. Furthermore, we isolated three other proteins from snake venoms that are homologous to ablomin and cloned the corresponding cDNAs. Two of these homologous proteins, triflin and latisemin, also inhibited high K+ -induced contraction of the artery. These results indicate that several snake venoms contain novel proteins with neurotoxin-like activity. [source] Purification, characterization, and cDNA cloning of a novel soluble saxitoxin and tetrodotoxin binding protein from plasma of the puffer fish, Fugu pardalisFEBS JOURNAL, Issue 22 2001Mari Yotsu-Yamashita Some species of puffer fish have been reported to possess both of tetrodotoxin and saxitoxin, which share one binding site on sodium channels. We purified a novel soluble glycoprotein that binds to these toxins from plasma of the puffer fish, Fugu pardalis, and named puffer fish saxitoxin and tetrodotoxin binding protein (PSTBP). PSTBP possessed a binding capacity of 10.6 ± 0.97 nmol·mg,1 protein and a Kd of 14.6 ± 0.33 nm for [3H]saxitoxin in equilibrium binding assays. [3H]Saxitoxin (10 nm) binding to PSTBPs was half-inhibited by the presence of tetrodotoxin and saxitoxin at 12 µm and 8.5 nm, respectively. From the results of gel filtration chromatography (200 kDa) and SDS/PAGE (104 kDa), PSTBP was suggested to consist of noncovalently linked dimers of a single subunit. PSTBP was completely deglycosylated by glycopeptidase F, producing a single band at 42 kDa. Two highly homologous cDNAs to each other coding PSTBP (PSTBP1 and PSTBP2, the predicted amino-acid identity 93%), were obtained from a cDNA library of F. pardalis liver. These proteins consisted to two tandemly repeated homologous domains. The predicted amino-acid sequences of PSTBP1 and 2 were not homologous to that of saxiphilin, a reported saxitoxin binding protein, or sodium channels, but their N-terminus sequences were homologous to that of the reported tetrodotoxin binding protein from plasma of Fugu niphobles, which has not been fully characterized. The partially homologous cDNA sequences to PSTBP1 and 2 were also found in expressed sequence tag clones of nontoxic flounders liver. Presumably, PSTBP is involved in accumulation and/or excretion of toxins in puffer fish. [source] Purification and cDNA cloning of nitric oxide reductase cytochrome P450nor (CYP55A4) from Trichosporon cutaneumFEBS JOURNAL, Issue 11 2001Li Zhang Cytochrome P450nor is involved in fungal denitrification as nitric oxide (NO) reductase. Although the heme protein has been known to occur in restricted species of fungi that belong to ascomycotina, we have previously suggested that it would also occur in the yeast Trichosporon cutaneum, which is phylogenetically far from those P450nor-producing ascomycetous fungi. Here we isolated and characterized the heme protein from the basidiomycetous yeast T. cutaneum. P450nor of the yeast (TcP450nor) exhibited properties in terms of catalysis, absorption spectrum and molecular mass that are almost identical to those of its counterparts in ascomycetous fungi. We also isolated and sequenced its cDNA. The predicted primary structure of TcP450nor showed high sequence identities (around 65%) to those of other P450nors, indicating that they belong to the same family. TcP450nor protein cofractionated with cytochrome c oxidase by subcellular fractionation and its predicted primary structure contained an extension on its amino terminus that is characteristic of a mitochondrial-targeting signal, indicating that it is a mitochondrial protein like some of the isoforms of other fungi. On the other hand, TcP450nor was unique in that inducers such as nitrate, nitrite, or NO were not required for its production in the cells. The occurrence of P450nor across the subdivisions of eumycota suggests that P450nor and denitrification are distributed more universally among fungi than was previously thought. [source] A novel hemocyte-specific membrane protein of Sarcophaga (flesh fly)FEBS JOURNAL, Issue 17 2000Shohei Hori Extensive tissue remodeling takes place during metamorphosis of holometabolous insects. It has been shown that hemocytes play crucial roles in the recognition and elimination of apoptotic cells and larval tissue fragments produced during metamorphosis. We report the immunoaffinity purification, cDNA cloning, and characterization of a prepupal hemocyte membrane protein of Sarcophaga (flesh fly) with a molecular mass of 120 kDa. This protein is a novel type I transmembrane protein with 18 repeats of an epidermal growth factor-like domain in the predicted extracellular region. Expression of the protein was restricted exclusively to prepupal hemocytes. This protein is suggested to be a scavenger receptor for tissue remodeling. [source] cDNA cloning, heat shock regulation and developmental expression of the hsp83 gene in the Mediterranean fruit fly Ceratitis capitataINSECT MOLECULAR BIOLOGY, Issue 6 2006M. A. Theodoraki Abstract This report presents the cDNA cloning, heat shock regulation and developmental expression of the hsp90 gene homologue of the Mediterranean fruit fly Ceratitis capitata (medfly). The isolated cDNA contained the coding region, the 3,UTR and most of the 5,UTR of the medfly hsp90 homologue, which was named Cchsp83. The deduced CcHSP83 polypeptide contained all the highly conserved amino acid segments that characterize the cytosolic members of the HSP90 family. Genomic analysis showed that the Cchsp83 gene is unique and was mapped at the 94C division of the sixth polytene chromosome. The size of the Cchsp83 mRNA was found to be approximately 2.7 kb. The predicted molecular mass of the CcHSP83 protein was 81.4 kDa, while the apparent molecular weight estimated by SDS-PAGE was approximately 90 kDa. Phylogenetic analysis based on 14 insect HSP90 amino acid sequences was consistent with the known phylogeny at low taxonomic level. The Cchsp83 gene is constitutively expressed in all stages of medfly development and is induced from a low level to several-fold by heat, depending on the developmental stage. Heat shock induction begins at 30 °C, reaching a maximum between 35 and 41 °C. Cchsp83 RNA expression is highly regulated during embryonic development; however, the temporal fluctuations in RNA levels during embryogenesis were not followed by similar fluctuations in the levels of the protein. [source] cDNA cloning, functional expression and characterization of kynurenine 3-hydroxylase of Anopheles stephensi (Diptera: Culicidae)INSECT MOLECULAR BIOLOGY, Issue 5 2002M. Hirai Abstract Kynurenine 3-hydroxylase (K3H) is a NADPH-dependent flavin monooxygenase involved in the tryptophan pathway. Xanthurenic acid (XA) is a metabolite of this pathway and has recently been identified as a gamete activating factor (GAF) of the malarial parasite. We cloned K3H cDNA from Anopheles stephensi (AsK3H), because anopheline mosquitoes are a vector of the human malaria parasite, Plasmodium falciparum and the catalytic function of AsK3H in XA production. Recombinant AsK3H protein was expressed in Sf-9 cells using the baculovirus system and its enzymatic properties were characterized. The specific activities of crude cell lysate and affinity purified protein were 94.9 ± 6.2 and 865.6 ± 10.5 nmol/min/mg protein, respectively. The optimum pH of AsK3H was 7.0. Analysis of AsK3H gene expression using RT-PCR revealed that AsK3H was constitutively expressed in egg, larva, pupa and adult. [source] cDNA of an arylphorin-type storage protein from Pieris rapae with parasitism inducible expression by the endoparasitoid wasp Pteromalus puparumINSECT SCIENCE, Issue 3 2009Jia-Ying Zhu Abstract, This report presents the cDNA cloning of a storage protein, PraAry, from Pieris rapae and investigates its expression regulated by parasitism of an endoparasitoid wasp Pteromalus puparum. The full-length cDNA of PraAry is 2 270 nucleotides and contains a 2 121 nucleotide open reading frame encoding 707 amino acids with calculated molecular weights of approximately 83 kDa. Analysis of the primary protein sequence revealed that it possesses a signal peptide of 16 amino acids at the N-terminus and contains two highly conserved storage protein signature motifs. According to both phylogenetic analysis and the criteria for amino acid composition, PraAry belongs to the subfamily of arylphorin-type storage protein (1.42% methionine and 18.82% aromatic amino acids). Reverse transcription , polymerase chain reaction analysis indicated that the transcriptional level of PraAry mRNA in P. rapae pupae fat body is inducible in response to parasitism by P. puparum. [source] cDNA cloning of the polymeric immunoglobulin receptor of the marsupial Macropus eugenii (tammar wallaby)INTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 2 2002C. L. Taylor Summary cDNA encoding a marsupial polymeric immunoglobulin receptor (pIgR) was isolated from Macropus eugenii (tammar wallaby) mammary lymph node primarily by reverse transcriptase coupled polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR. This resulted in a 5, truncated clone and, in order to obtain the full-length sequence, genomic walking PCR was utilized. The complete sequence consists of 2696 bp of cDNA and encodes a predicted polypeptide of 732 amino acids. The wallaby sequence is highly conserved in relation to the only other reported marsupial pIgR sequence, that of Trichosurus vulpecula (brushtail possum), having a nucleotide identity of 86.7% and a deduced amino acid identity of 79.9%. The wallaby nucleotide sequence also has a moderate degree of similarity with the pIgR sequences of eutherian mammals, being most similar to that of the rat, with an identity of 63.1%. At the amino acid level, in comparison to eutherian sequences, the wallaby pIgR is most similar to that of humans with an identity of 52.6%. pIgR phylogenetic trees were constructed for tammar wallaby, brushtail possum and several eutherian mammal cDNA and deduced amino acid sequences. In both DNA and protein analyses, the eutherian sequences formed a sister clade to the exclusion of the marsupial sequences, in agreement with the current view of mammalian evolution. [source] Purification and characterization of a new reovirus from the Chinese mitten crab, Eriocheir sinensisJOURNAL OF FISH DISEASES, Issue 12 2004S Zhang Abstract A new reovirus was recently isolated from a freshwater crab, the Chinese mitten crab, Eriocheir sinensis, in China. The complete viral particles are 55 nm in diameter, icosahedral, non-enveloped and have a mean buoyant density of 1.39 g cm,3 in CsCl gradient. The viral genome is composed of 12 pieces of dsRNA with an electrophoretic pattern of 3/4/2/3. This virus infects connective tissue of the gills, gut and hepatopancreas. Partial cDNA cloning and sequence analysis showed that the RNA-dependent RNA polymerase is located in the first RNA segment. From its biochemical, ultrastructural and physicochemical properties, this virus is quite different from the genus Aquareovirus (Reoviridae). It may represent a new genus of Reoviridae, different from the other crab reoviruses, P and W2. [source] Two tachykinin-like peptides from skin secretions of Danio rerioJOURNAL OF PEPTIDE SCIENCE, Issue 2 2010Xuhua Mi Abstract Tachykinin perform multiple physiological functions such as smoothing muscle contraction, vasodilation, inflammation, the processing of nerve signal, neuroprotection and neurodegeneration. Two novel tachykinin-like peptides named tachykinin-DR1 and -DR2 were identified from skin secretions of Danio rerio in current work. Their amino acid sequences were determined as SKSQHFHGLM-NH2 and NKGEIFVGLM-NH2, respectively. They share a conserved FXGLM-NH2C -terminal consensus motif. By cDNA cloning, the precursor encoding both tachykinin-DR1 and -DR2 was screened from the skin cDNA library of D. rerio. Tachykinin-DR1 and -DR2 share the same precursor, which is composed of 108 amino acid (aa) residues. Regarding the biological activity, tachykinin-DRs could induce the contraction of isolated strips of guinea pig ileum just like other tackykinins. To our best knowledge, this is the first report of tachykinin from fish skin. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd. [source] cDNA cloning of the housefly pigment-dispersing factor (PDF) precursor protein and its peptide comparison among the insect circadian neuropeptidesJOURNAL OF PEPTIDE SCIENCE, Issue 2 2004Ayami Matsushima Abstract Pigment-dispersing factor (PDF), an 18-amino acid neuropeptide, is a principal circadian neurotransmitter for the circadian rhythms of the locomotor activity in flies. Recently, two completely different types of PDF precursor were clarified; that of the cricket Gryllus bimaculatus and that of the last-summer cicada Meimuna opalifera. The G. bimaculatus PDF precursor is extraordinarily short and comprises a nuclear localization signal (NLS), while the M. opalifera PDF precursor is of ordinary length, comparable to that seen for the precursors of crustacean ,-PDH homologues. Although their PDF peptide regions were exactly the same, the regions containing a signal peptide combined with a PDF-associated peptide (PAP) were remarkably different from each other. Such a grouping suggested a fundamental role for the PAP peptide in the circadian clock, perhaps associated with PDF function. In the present study, the cDNA cloning of PDF from the adult brains of the housefly Musca domestica was carried out and it was found that an isolated clone (527 bp) encodes a PDF precursor protein of ordinary length. The PDF peptide shows a high sequence identity (78%,94%) and similarity (89%,100%) to insect PDFs and also to the crustacean ,-PDH peptides. In particular, there is only a single amino acid difference between the PDFs of Musca and Drosophila; at position 14 Ser for Musca PDF and Asn for Drosophila PDF. A characteristic Ser10 in Drosophila was retained in Musca, indicating the presence of a structural profile unique to these PDFs. The results of sequence analyses suggest that Musca and Drosophila PDFs are to be considered members of a single group that has evolved structurally. When the primary structure of the PAP regions was compared, the Musca PDF precursor also belonged to the same group as that to which the Drosophila PDF precursor belongs. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd. [source] Identification of testis-specific ubiquitin-conjugating enzyme in the ascidian Ciona intestinalisMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 7 2010Naoto Yokota The ubiquitin,proteasome system is known to play a key role in fertilization in ascidians, sea urchins, and mammals. To obtain insights into the ubiquitin-conjugating enzymes (Ube2) involved in reproductive systems, we systematically explored Ube2 enzymes expressed in the testis of the ascidian Ciona intestinalis. Here, we report cDNA cloning and characterization of a novel type of Ube2r (Ci0100152677) that is capable of making a thiolester bond with ubiquitin. Northern analysis, whole-mount in situ hybridization and immunocytochemistry indicate that this enzyme is exclusively expressed in the testis, mainly in the germ cells during the late stage of spermatogenesis, and is localized in the sperm head and tail, suggesting possible participation in fertilization or spermatogenesis/spermiogenesis. Mol. Reprod. Dev. 77: 640,647, 2010. © 2010 Wiley-Liss, Inc. [source] cDNA cloning of a lectin-like gene preferentially expressed in freshwater from the macroalga Ulva limnetica (Ulvales, Chlorophyta)PHYCOLOGICAL RESEARCH, Issue 2 2009Kensuke Ichihara SUMMARY The macroalgal species Ulva limnetica Ichihara et Shimada was investigated to understand the molecular mechanism of its tolerance or adaptation to freshwater. We detected a 19 kDa protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which accumulated in greater amounts in freshwater conditions compared with marine conditions. The band was excised and the partial amino acid sequence was determined by Edoman degradation. Based on the sequences, we isolated the corresponding cDNA by the rapid amplification of cDNA ends (RACE) technique. The constructed, full-length cDNA was 1272 bp in length, consisting of 198 bp 5,-non-coding region, an open reading frame of 840 bp (279 amino acids), 233 bp 3,-non-coding region and poly (A) tail. The protein encoded by the cDNA showed 30% identity and 45% similarity to lectin isolated from Ulva pertusa Kjellman, and we named this gene ULL (encoding Ulva limnetica lectin-like protein). Northern blot analysis demonstrated that the expression level of the ULL in the freshwater-cultured sample was higher than in the seawater-cultured sample. [source] The fallaxidin peptides from the skin secretion of the Eastern Dwarf Tree Frog Litoria fallax.RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 20 2008Sequence determination by positive, cDNA cloning of the fallaxidins, negative ion electrospray mass spectrometry: antimicrobial activity The glandular skin secretion of the Eastern Dwarf Tree Frog Litoria fallax contains nine peptides named fallaxidins. The sequences of these peptides were elucidated using a combination of positive and negative electrospray mass spectrometry together with Edman sequencing. Among these peptides are: (i) fallaxidins 1.1 and 2.1 which have the sequences YFPIPI-NH2 and FWPFM-NH2. The activities of these peptides are unknown, but it has been shown that they are not smooth muscle active, opioids or antimicrobially active, nor do they effect proliferation of lymphocytes; (ii) two weakly active antibiotics, fallaxidins 3.1 and 3.2 (e.g. fallaxidin 3.1, GLLDLAKHVIGIASKL-NH2), and a moderately active antibiotic fallaxidin 4.1 (GLLSFLPKVIGVIGHLIHPPS-OH). Fallaxidin 4.1 has an unusual sequence for an antibiotic, containing three Pro residues together with a C-terminal CO2H group. cDNA cloning has confirmed the identity of the nine isolated peptides from L. fallax, together with five additional peptides not detected in the peptide profile. The pre-regions of the nine preprofallaxidins are conserved and similar to those of the caerin peptides from L. caerulea and L. splendida, suggesting that the fallaxidin and caerin peptides, although significantly different in sequence, originated from a common ancestor gene. Copyright © 2008 John Wiley & Sons, Ltd. [source] Identification and characterization of a novel anthocyanin malonyltransferase from scarlet sage (Salvia splendens) flowers: an enzyme that is phylogenetically separated from other anthocyanin acyltransferasesTHE PLANT JOURNAL, Issue 6 2004Hirokazu Suzuki Summary Anthocyanin acyltransferases (AATs) catalyze a regiospecific acyl transfer from acyl-CoA to the glycosyl moiety of anthocyanins, thus playing an important role in flower coloration. The known AATs are subfamily members of an acyltransferase family, the BAHD family, which play important roles in secondary metabolism in plants. Here, we describe the purification, characterization, and cDNA cloning of a novel anthocyanin malonyltransferase from scarlet sage (Salvia splendens) flowers. The purified enzyme (hereafter referred to as Ss5MaT2) is a monomeric 46-kDa protein that catalyzes the transfer of the malonyl group from malonyl-CoA to the 4,,,-hydroxyl group of the 5-glucosyl moiety of anthocyanins. Thus, it is a malonyl-CoA:anthocyanin 5-glucoside 4,,,- O -malonyltransferase. On the basis of the partial amino acid sequences of the purified enzyme, we isolated a cDNA that encodes an acyltransferase protein. The steady-state transcript level of the gene was the highest in recently opened, fully pigmented flowers and was also correlated with the trend observed for an AAT gene responsible for the first malonylation step during salvianin biosynthesis. Immunoprecipitation studies using antibodies against the recombinant acyltransferase protein corroborated the identity of this cDNA as that encoding Ss5MaT2. The deduced amino acid sequence of Ss5MaT2 showed a low similarity (22,24% identity) to those of AATs and lacked the AAT-specific signature sequence. A phylogenetic analysis suggested that Ss5MaT2 is more related to acetyl-CoA:benzylalcohol acetyltransferase (BEAT) rather than to AAT. This is another example in which enzymes with similar, although not identical, substrate evolved from different branches of the BAHD family. [source] cDNA cloning, genomic structure and polymorphism of the porcine FHL3 geneANIMAL GENETICS, Issue 3 2004B. Zuo Summary LIM domain proteins are important regulators of the growth, determination and differentiation of cells. Four-and-a-half LIM-only protein 3 (FHL3) is a type of LIM-only protein that contains four tandemly repeated LIM motifs with an N-terminal single zinc finger (half LIM motif). In this study, we have determined the complete coding sequence of pig FHL3 which encodes a 280 amino acid protein. The coding region of the pig FHL3 gene is organized in five exons and spans an approximately 2.1-kb genomic region. Comparative sequencing of six pig breeds revealed three single nucleotide polymorphisms (SNPs) within exon 2 of which an A,G substitution at position 313 changes a codon for arginine into a codon for glycine. The substitution was situated within a PstI recognition site and developed as a PCR-RFLP marker for further use in population variation investigations and association analysis. The A/G polymorphism was segregating only in Landrace pigs. Association studies of the FHL3 polymorphism with carcass traits provided preliminary evidence that the PstI PCR-restriction fragment length polymorphism (RFLP) genotype may be associated with variation in several carcass traits of interest for pig breeding. Further investigations in more Landrace pigs are needed to confirm this. [source] cDNA cloning and genetic polymorphism of the swine major histocompatibility complex (SLA) class II DMA geneANIMAL GENETICS, Issue 2 2001A. Ando cDNA clones corresponding to the swine histocompatibility complex (SLA: swine leucocyte antigen)-DM , chain were isolated using the polymerase chain reaction (PCR) products from the third exon in the human HLA-DMA gene as a probe. Amino acid comparative analysis revealed that these clones were more closely related to the bovine and human DMA genes than to the other swine class II genes , chain genes, DRA, DQA and DOA. These results suggest that the SLA-DMA gene is expressed and may function, like HLA-DM, as an important modulator in class II restricted antigen processing in swine. Furthermore, based on the sequences and PCR,restriction fragment length polymorphism (PCR,RFLP) patterns in the SLA-DMA gene, no allelic variation was recognized in the second exon, but five allelic variations were recognized in the third exon in five different breeds of swine. These DMA alleles were defined by variation at four nucleotide positions. Two of these alleles resulted in an amino acid substitution. These results suggest that SLA-DMA has little polymorphism as observed in HLA-DMA and mouse H2-Ma. [source] cDNA cloning and expression analysis of the myosin heavy chain (MYH) gene of the mandarin fish Siniperca kneriAQUACULTURE RESEARCH, Issue 4 2009Jianshe Zhang Abstract In this study, we applied RT-PCR and cDNA cloning techniques to clone myosin heavy chain (MYH) cDNA from muscle tissues of the mandarin fish Siniperca kneri. The cDNA was determined to be of 6987 base pairs in length, encoding a peptide of 1937 amino acids (Genbank accession no. EF446616). A search of encoded protein sequences in the NCBI conserved domain database indicated the presence of all known protein domains for MYH proteins, i.e. the myosin motor domain in the N-terminal region, the DIL domain at the C-terminus, and the ATPase domain. The MYH gene and its protein were expressed predominantly in muscle tissues and weakly in cardiac tissues. Developmentally, the MYH gene was first expressed in the muscle formation stage and continued later on. Our work provided a novel mypsin heavy chain gene sequence in fish biology and the results indicate that the MYH gene and the protein it encodes are important for the growth and development of the mandarin fish, as well as its muscle characterization. [source] cDNA cloning and induction of tyrosine hydroxylase gene from the diamondback moth, Plutella xylostellaARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2010Se Hui Hwang Abstract We cloned a full-length tyrosine hydroxylase cDNA from the integument of the diamondback moth, Plutella xylostella. In the phylogenetic tree, tyrosine hydroxylase (PxTH) clustered with the other lepidopteran THs. Serine residues in the PxTH sequence, namely Ser24, Ser31, Ser35, Ser53, and Ser65, were predicted to be the target sites for phosphorylation based on PROSITE analysis. In particular, Ser35 of PxTH is highly conserved across a broad phylogenetic range of animal taxa including rat and human. Western blot analysis using both PxTH-Ab1 and PxTH-Ab2 polyclonal antibodies verified the expression of PxTH in all life cycle stages of P. xylostella, namely the larval, pupal, and adult stages. To examine the possible immune function of PxTH in P. xylostella, PxTH gene expression was investigated by RT-PCR and western blotting analysis after challenging P. xylostella with bacteria. PxTH expression was elevated 1,h post-infection and was continued till 12,h of post-infection relative to control larvae injected with sterile water. © 2010 Wiley Periodicals, Inc. [source] Cloning, expression and purification of cytochrome c6 from the brown alga Hizikia fusiformis and complete X-ray diffraction analysis of the structureACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2008Hideharu Akazaki The primary sequence of cytochrome c6 from the brown alga Hizikia fusiformis has been determined by cDNA cloning and the crystal structure has been solved at 1.6,Å resolution. The crystal belonged to the tetragonal space group P41212, with unit-cell parameters a = b = 84.58, c = 232.91,Å and six molecules per asymmetric unit. The genome code, amino-acid sequence and crystal structure of H. fusiformis cytochrome c6 were most similar to those of red algal cytochrome c6. These results support the hypothesis that brown algae acquired their chloroplasts via secondary endosymbiosis involving a red algal endosymbiont and a eukaryote host. [source] | ||||||||||||||||