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Rapid Amplification (rapid + amplification)
Selected AbstractsLAF4, an AF4 -related gene, is fused to MLL in infant acute lymphoblastic leukemiaGENES, CHROMOSOMES AND CANCER, Issue 1 2002Anne R.M. von Bergh Infant acute lymphoblastic leukemia (ALL) with MLL gene rearrangements is characterized by a proB phenotype and a poor clinical outcome. We analyzed an infant proB ALL with t(2;11)(p15;p14) and an MLL rearrangement on Southern blot analysis. Rapid amplification of cDNA ends,polymerase chain reaction (PCR) and reverse transcriptase-PCR identified the LAF4 gene mapped on chromosome region 2q11.2,q12 as a fusion partner of the MLL gene. The LAF4 gene was identified previously by its high sequence homology to the AF4 protein and encodes a protein of 1,227 amino acids. The t(4;11)(q21;q23), creating the MLL - AF4 chimeric transcripts, is the predominant 11q23 chromosome translocation in infant ALL and is associated with an extremely poor prognosis. Our findings further suggest that fusion of MLL to one of the AF4 family members (AF4/LAF4/AF5Q31) might determine a proB-cell phenotype in infant leukemia. © 2002 Wiley-Liss, Inc. [source] Rapid amplification and cloning of Tn5 flanking fragments by inverse PCRLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2000G. Huang A simple approach is described to efficiently amplify DNA sequences flanking transposon Tn5 insertions. The method involves: (i) digestion with a restriction enzyme that cuts within Tn5; (ii) self-ligation under conditions favouring the production of monomeric circles; (iii) four parallel PCR reactions using primers designed to amplify left or right flanking sequences, and to distinguish target amplicons from non-specific products. This reveals the number of Tn5 insertions and the size of flanking genomic restriction fragments, without Southern blot analysis. The amplified product contains restriction sites that facilitate cohesive-end cloning. This rapid method is demonstrated using Tn5 and Tn5-Mob tagged DNA sequences involved in albicidin biosynthesis in Xanthomonas albilineans. It is generally applicable for efficient recovery of DNA sequences flanking transposon Tn5 derivatives in insertional mutagenesis studies. [source] Coordinated and conserved expression of alphoid repeat and alphoid repeat-tagged coding sequencesDEVELOPMENTAL DYNAMICS, Issue 1 2003Yin-Xiong Li Abstract We have found an alpha-like simple-sequence DNA repeat that is differentially expressed during early embryogenesis in both chick and zebrafish. Before and during the primitive streak stage, transcripts of the alphoid repeat sequence were ubiquitously expressed throughout zebrafish and chick embryos. After headfold formation, expression was limited to the cardiac neural crest, the head, and the heart. Two types of alphoid repeat sequence transcripts were identified: alphoid repeat RNA and alphoid repeat-tagged mRNA (ES,T). Several of the ES,Ts were identified by (1) searching expressed sequence tag databases, (2) arbitrary rapid amplification of cDNA ends (RACE), and (3) screening embryonic cDNA libraries. The alphoid element was located in the 3, untranslated region of one ES,T that was obtained by RACE. The ES,T sequences encoded a variety of different types of proteins, but all were expressed within tissues that were positive for the alphoid repeat RNA. The presence of two types of coordinately expressed alphoid-like repeat transcripts in maternal RNA with subsequent restriction to the head and heart, and the conservation of these features in disparate vertebrate embryos, suggest that the alphoid repeat sequence may serve as a control element in the gene regulation network. Developmental Dynamics 228:72,81, 2003. © 2003 Wiley-Liss, Inc. [source] Localization of RNAs in oocytes of Eleutherodactylus coqui, a direct developing frog, differs from Xenopus laevisEVOLUTION AND DEVELOPMENT, Issue 6 2003Yvonne M. Beckham SummaryEleutherodactylus coqui develops directly on land to a frog. The large 3.5-mm oocyte of E. coqui has enough yolk to allow development without a feeding tadpole. In the smaller Xenopus laevis oocyte, 1.3 mm in diameter, mRNAs involved in germ layer formation, such as VegT and Vg1, are localized to the vegetal cortex of the oocyte. We hypothesized that an animal shift has occurred in the localization of the E. coqui Orthologs of VegT and Vg1 due to the large egg size. Through a combination of degenerate reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), we cloned 1634 bp of EcVegT and 1377 bp of EcVg1. Northern blot analysis shows that the lengths of these transcripts are 2.5 kb and 1.3 kb, respectively. This result suggests that we have obtained the complete Vg1 transcript, although this transcript has an extremely short 3, untranslated region compared with X. laevis, 256 bp and 1268 bp, respectively. Zygotic expression of EcVegT closely resembles that of VegT, supporting their orthology. Radioactive RT-PCR and in situ hybridization demonstrated the presence of EcVegT and EcVg1 predominantly near the animal pole of the oocyte. RT-PCR showed that the animal blastomeres, formed from the first horizontal cleavage, inherit half of the EcVegT and EcVg1 transcripts, although they contain only about 1% of the embryo volume. Our results indicate major differences between the molecular organization of the eggs of X. laevis and E. coqui. [source] Characterization and gene cloning of a novel serine protease with nematicidal activity from Trichoderma pseudokoningii SMF2FEMS MICROBIOLOGY LETTERS, Issue 2 2009Lei-Lei Chen Abstract Trichoderma pseudokoningii SMF2 is a biocontrol fungus with inhibitory ability against phytopathogenic fungi. Here, a crude extract of strain SMF2 in a solid ferment exhibited strong nematicidal activity against Meloidogyne incognita, and a novel serine protease SprT with nematicidal activity was purified from the crude extract. Protease SprT has a molecular mass of 31 kDa, a pH optimum of 8.5, and a temperature optimum of 60,65 °C. It had good thermostability, and was stable in an alkaline environment. SprT could degrade bovine serum albumin, lysozyme, and gelatin, and its activity was enhanced by many metal ions. The cuticles of nematodes treated by protease SprT obviously crimpled. Purified protease SprT could kill juveniles of M. incognita and inhibit egg hatch, suggesting that it is involved in the nematicidal process of T. pseudokoningii SMF2. The full-length cDNA gene-encoding protease SprT was cloned by rapid amplification of cDNA ends. Sequence analysis showed that SprT is a monodomain subtilase containing 284 amino acid residues. It had higher identities and a closer relation to the nematicidal serine proteases (59,69%) from nematode parasitic fungi than to the serine proteases (<50%) from Trichoderma. Protease SprT represents the first well-characterized subtilase with nematicidal activity from Trichoderma. [source] Cloning and expression of a geranylgeranyl diphosphate synthase gene: insights into the synthesis of termite defence secretionINSECT MOLECULAR BIOLOGY, Issue 1 2007Masaru Hojo Abstract In Nasutitermes takasagoensis, a termite in which soldiers perform specialized chemical defence, Nts19-1 gene is highly expressed exclusively in soldier head. In this study, two types of transcripts for this gene were obtained, and the full-length cDNAs were determined by rapid amplification of cDNA ends (RACE). These transcripts were putative homologues of the geranylgeranyl diphosphate (GGPP) synthase gene, involved in the condensation of dimethylallyl diphosphate with isopentenyl diphosphate in the isoprenoid biosynthetic pathway. The genes were thus termed NtGGPPS1. GGPP is a precursor of diterpenes in plants. In situ hybridization localized NtGGPPS1 expression to the epidermal secretory cells of the frontal gland reservoir where many kinds of diterpenes are produced, suggesting that NtGGPPS1 is involved in the biosynthesis of defence secretion. [source] Partial genomic organization of ribosomal protein S7 gene from malaria vector Anopheles stephensiINSECT SCIENCE, Issue 2 2007RAJNIKANT DIXIT Abstract In this study, we describe the partial genomic organization of ribosomal protein S7 gene isolated from the mosquito Anopheles stephensi. Initially a 558 bp partial cDNA sequence was amplified as precursor mRNA sequence containing 223 bp long intron. 5, and 3, end sequences were recovered using end specific rapid amplification of cDNA ends (RACE) polymerase chain reaction. The full-length cDNA sequence was 914 nucleotide long with an open reading frame capable of encoding 192 amino acid long protein with calculated molecular mass of 22 174 Da and a pI point of 9.94. Protein homology search revealed > 75% identity to other insect's S7 ribosomal proteins. Analysis of sequence alignment revealed several highly conserved domains, one of which is related to nuclear localization signal (NLS) region of human rpS7. Interestingly, intron nucleotide sequence comparison with A. gambiae showed a lesser degree of conservation as compared to coding and untranslated regions. Like this, early studies on the genomic organization and cDNA/ Expressed sequence tag analysis (EST) could help in genome annotation of A. stephensi, and would be likely to be sequenced in the future. [source] An interferon-sensitive response element is involved in constitutive caspase-8 gene expression in neuroblastoma cellsINTERNATIONAL JOURNAL OF CANCER, Issue 1 2007Alessandro De Ambrosis Abstract We previously identified a 1.2 Kb DNA element (P-1161/+16), 5, to caspase-8 exon-1, that acts as promoter in caspase-8-positive, but not in caspase-8-negative neuroblastoma (NB) cells. The P-1161/+16 DNA element regulates both constitutive and interferon IFN-,-inducible caspase-8 expression. Two GAS (IFN-activated sequence, STAT-1 binding site) and two ISRE (interferon sensitive response element, IRF binding site) were present in P-1161/+16. Deletion studies indicated that elements essential for promoter activity in NB cells were present in a 167 bp region 5, flanking exon-1 (P-151/+16), which contains an ISRE at position ,32. The transcription initiation site was mapped by 5, rapid amplification of cDNA end (RACE) at position ,20 from caspase-8 cDNA reference sequence. Disruption of the ISRE-32 indicated that it is required for both constitutive and IFN-,-inducible caspase-8 expression. IRF-1 and IRF-2 transcription factors bind to the (,151/+16) DNA fragment in vitro. Chromatin immunoprecipitation (ChIP) assays showed that IRF-1 and IRF-2 bind to the DNA region at the 5, of caspase-8 gene in NB cells, which show constitutive expression but not in caspase-8 negative cells. In these last cells, up-regulation of caspase-8 by IFN-, was associated to induction of IRF-1 and IRF-2 binding to caspase-8 promoter and increased histone acetylation. Moreover, RNA interference experiments also supported the involvement of IRF-1 and IRF-2 in constitutive caspase-8 expression in NB cells. © 2006 Wiley-Liss, Inc. [source] Identification of a novel human tissue factor splice variant that is upregulated in tumor cells,INTERNATIONAL JOURNAL OF CANCER, Issue 7 2006Hitendra S. Chand Abstract Tissue factor (TF) is a transmembrane glycoprotein that serves as the prime initiator of blood coagulation and plays a critical role in thrombosis and hemostasis. In addition, a variety of tumor cells overexpress cell-surface TF, which appears to be important for tumor angiogenesis and metastasis. To elucidate the mechanism involved in the upregulation of TF in human tumor cells, a comprehensive analysis of TF mRNA from various normal and tumor cells was performed. The results of these studies indicate that, in addition to possessing a normal full-length TF transcript and minor levels of an alternatively spliced transcript known as alternatively-spliced tissue factor (asTF) (Bogdanov et al., Nat Med 2003;9:458,62), human tumor cells express additional full-length TF transcripts that are also generated by alternative splicing. Reverse transcriptase-polymerase chain reaction (RT-PCR) and 5,-rapid amplification of cDNA ends- (5,-RACE) based analyses of cytoplasmic RNA from normal and tumor cells revealed that there is alternative splicing of the first intron between exon I and exon II resulting in 2 additional TF transcripts. One of the transcripts has an extended exon I with inclusion of most of the first TF intron (955 bp), while the second transcript is formed by the insertion of a 495 bp sequence, referred to as exon IA, derived from an internal sequence of the first intron. The full length TF transcript with alternatively spliced novel exon IA, referred to as alternative exon 1A-tissue factor (TF-A), represented ,1% of the total TF transcripts in normal cells, but constituted 7,10% of the total TF transcript in tumor cells. Quantitative real-time RT-PCR analysis indicated that cultured human tumor cells contain 10,25-fold more copy numbers of TF-A in comparison to normal, untransformed cells. We propose that high-level expression of the novel TF-A transcript, preferentially in tumor cells, may have utility in the diagnosis and staging of a variety of solid tumors. © 2005 Wiley-Liss, Inc. [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] 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] Purification of Matrix Gla Protein From a Marine Teleost Fish, Argyrosomus regius: Calcified Cartilage and Not Bone as the Primary Site of MGP Accumulation in Fish,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2003DC Simes Abstract Matrix Gla protein (MGP) belongs to the family of vitamin K-dependent, Gla-containing proteins, and in mammals, birds, and Xenopus, its mRNA was previously detected in extracts of bone, cartilage, and soft tissues (mainly heart and kidney), whereas the protein was found to accumulate mainly in bone. However, at that time, it was not evaluated if this accumulation originated from protein synthesized in cartilage or in bone cells because both coexist in skeletal structures of higher vertebrates and Xenopus. Later reports showed that MGP also accumulated in costal calcified cartilage as well as at sites of heart valves and arterial calcification. Interestingly, MGP was also found to accumulate in vertebra of shark, a cartilaginous fish. However, to date, no information is available on sites of MGP expression or accumulation in teleost fishes, the ancestors of terrestrial vertebrates, who have in their skeleton mineralized structures with both bone and calcified cartilage. To analyze MGP structure and function in bony fish, MGP was acid-extracted from the mineralized matrix of either bone tissue (vertebra) or calcified cartilage (branchial arches) from the bony fish, Argyrosomus regius,, separated from the mineral phase by dialysis, and purified by Sephacryl S-100 chromatography. No MGP was recovered from bone tissue, whereas a protein peak corresponding to the MGP position in this type of gel filtration was obtained from an extract of branchial arches, rich in calcified cartilage. MGP was identified by N-terminal amino acid sequence analysis, and the resulting protein sequence was used to design specific oligonucleotides suitable to amplify the corresponding DNA by a mixture of reverse transcription-polymerase chain reaction (RT-PCR) and 5,rapid amplification of cDNA (RACE)-PCR. In parallel, ArBGP (bone Gla protein, osteocalcin) was also identified in the same fish, and its complementary DNA cloned by an identical procedure. Tissue distribution/accumulation was analyzed by Northern blot, in situ hybridization, and immunohistochemistry. In mineralized tissues, the MGP gene was predominantly expressed in cartilage from branchial arches, with no expression detected in the different types of bone analyzed, whereas BGP mRNA was located in bone tissue as expected. Accordingly, the MGP protein was found to accumulate, by immunohistochemical analysis, mainly in the extracellular matrix of calcified cartilage. In soft tissues, MGP mRNA was mainly expressed in heart but in situ hybridization, indicated that cells expressing the MGP gene were located in the bulbus arteriosus and aortic wall, rich in smooth muscle and endothelial cells, whereas no expression was detected in the striated muscle myocardial fibers of the ventricle. These results show that in marine teleost fish, as in mammals, the MGP gene is expressed in cartilage, heart, and kidney tissues, but in contrast with results obtained in Xenopus and higher vertebrates, the protein does not accumulate in vertebra of non-osteocytic teleost fish, but only in calcified cartilage. In addition, our results also indicate that the presence of MGP mRNA in heart tissue is due, at least in fish, to the expression of the MGP gene in only two specific cell types, smooth muscle and endothelial cells, whereas no expression was found in the striated muscle fibers of the ventricle. In light of these results and recent information on expression of MGP gene in these same cell types in mammalian aorta, it is likely that the levels of MGP mRNA previously detected in Xenopus, birds, and mammalian heart tissue may be restricted toregions rich in smooth muscle and endothelial cells. Our results also emphasize the need to re-evaluate which cell types are involved in MGP gene expression in other soft tissues and bring further evidence that fish are a valuable model system to study MGP gene expression and regulation. [source] W55a Encodes a Novel Protein Kinase That Is Involved in Multiple Stress ResponsesJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 1 2009Zhao-Shi Xu Abstract Protein kinases play crucial roles in response to external environment stress signals. A putative protein kinase, W55a, belonging to SNF1-related protein kinase 2 (SnRK2) subfamily, was isolated from a cDNA library of drought-treated wheat seedlings. The entire length of W55a was obtained using rapid amplification of 5, cDNA ends (5,-RACE) and reverse transcription-polymerase chain reaction(RT-PCR). It contains a 1 029 -bp open reading frame (ORF) encoding 342 amino acids. The deduced amino acid sequence of W55a had eleven conserved catalytic subdomains and one Ser/Thr protein kinase active-site that characterize Ser/Thr protein kinases. Phylogenetic analysis showed that W55a was 90.38% homologous with rice SAPK1, a member of the SnRK2 family. Using nullisomic-tetrasomic and ditelocentric lines of Chinese Spring, W55a was located on chromosome 2BS. Expression pattern analysis revealed that W55a was upregulated by drought and salt, exogenous abscisic acid, salicylic acid, ethylene and methyl jasmonate, but was not responsive to cold stress. In addition, W55a transcripts were abundant in leaves, but not in roots or stems, under environmental stresses. Transgenic Arabidopsis plants overexpressing W55a exhibited higher tolerance to drought. Based on these findings, W55a encodes a novel dehydration-responsive protein kinase that is involved in multiple stress signal transductions. [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] Research note: Characterization of a cDNA encoding glutamine synthetase II from Gelidium crinale (Rhodophyta)PHYCOLOGICAL RESEARCH, Issue 1 2002D. Wilson Freshwater SUMMARY A cDNA encoding glutamine synthetase (GS) was characterized from the red alga, Gelidium crinale (Turner) Gaillon, using reverse-transcriptase polymerase chain reaction and the 5,- and 3,-rapid amplification of cDNA ends. Sequence analysis of a 1231-bp GS cDNA transcript included both 5, and 3, untranslated regions and a 1056-bp open reading frame encoding a 352 amino acid polypeptide. Comparison with GS sequences from other organisms revealed that the G. crinale cDNA encodes a type-II GS, and the absence of a N-terminal plastid signal sequence suggests that it is a cytosolic isoenzyme. Phylogenetic analyses of GSII amino acid sequences supports the multiple origin of cytosolic and plastid isoenzymes during eukaryotic evolution. [source] Detection of Strawberry crinkle virus in plants and aphids by RT-PCR using conserved L gene sequencesPLANT PATHOLOGY, Issue 3 2002K. I. Posthuma About 10% of the large (L) protein gene of Strawberry crinkle virus (SCV) was sequenced after amplification with degenerate primers designed to conserved regions of the rhabdovirus L protein. The virus sequence was extended to 1362 nucleotides through rapid amplification of cDNA ends. One pair of degenerate L gene primers amplified a 683-bp fragment from four different isolates of SCV cultured in the experimental host Physalis pubescens; the nucleotide sequences of these fragments differed by < 1% to 10% indicating the suitability of this region as a diagnostic target. This information enabled the development of a reverse transcription polymerase chain reaction (RT-PCR) detection method for SCV using primers designed to the L gene sequence. SCV was amplified from infected P. pubescens (573 bp fragment) and from infected Chaetosiphon fragaefolii aphids (770 bp fragment). SCV was also detected by RT-PCR in total RNA extracts from three strawberry plants showing symptoms typical of SCV infection but failed when the intensity of the disease symptoms decreased. However, both SCV positive-sense RNA, and negative-sense genomic RNA, were detected by nested PCR in chronically infected strawberry plants sampled in September. [source] Two novel neuropeptides in innervation of the salivary glands of the black-legged tick, Ixodes scapularis: Myoinhibitory peptide and SIFamideTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 5 2009Ladislav The peptidergic signaling system is an ancient cell,cell communication mechanism that is involved in numerous behavioral and physiological events in multicellular organisms. We identified two novel neuropeptides in the neuronal projections innervating the salivary glands of the black-legged tick, Ixodes scapularis (Say, 1821). Myoinhibitory peptide (MIP) and SIFamide immunoreactivities were colocalized in the protocerebral cells and their projections terminating on specific cells of salivary gland acini (types II and III). Immunoreactive substances were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis: a 1,321.6-Da peptide with the sequence typical for MIP (ASDWNRLSGMWamide) and a 1,395.7-Da SIFamide (AYRKPPFNGSIFamide), which are highly conserved among arthropods. Genes encoding these peptides were identified in the available Ixodes genome and expressed sequence tag (EST) database. In addition, the cDNA encoding the MIP prepropeptide was isolated by rapid amplification of cDNA ends (RACE). In this report, we describe the anatomical structure of specific central neurons innervating salivary gland acini and identify different neuropeptides and their precursors expressed by these neurons. Our data provide evidence for neural control of salivary gland by MIP and SIFamide from the synganglion, thus lending a basis for functional studies of these two distinct classes of neuropeptides. J. Comp. Neurol. 517:551,563, 2009. © 2009 Wiley-Liss, Inc. [source] Two novel neuropeptides in innervation of the salivary glands of the black-legged tick, Ixodes scapularis: Myoinhibitory peptide and SIFamideTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 5 2009Ladislav Abstract The peptidergic signaling system is an ancient cell,cell communication mechanism that is involved in numerous behavioral and physiological events in multicellular organisms. We identified two novel neuropeptides in the neuronal projections innervating the salivary glands of the black-legged tick, Ixodes scapularis (Say, 1821). Myoinhibitory peptide (MIP) and SIFamide immunoreactivities were colocalized in the protocerebral cells and their projections terminating on specific cells of salivary gland acini (types II and III). Immunoreactive substances were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis: a 1,321.6-Da peptide with the sequence typical for MIP (ASDWNRLSGMWamide) and a 1,395.7-Da SIFamide (AYRKPPFNGSIFamide), which are highly conserved among arthropods. Genes encoding these peptides were identified in the available Ixodes genome and expressed sequence tag (EST) database. In addition, the cDNA encoding the MIP prepropeptide was isolated by rapid amplification of cDNA ends (RACE). In this report, we describe the anatomical structure of specific central neurons innervating salivary gland acini and identify different neuropeptides and their precursors expressed by these neurons. Our data provide evidence for neural control of salivary gland by MIP and SIFamide from the synganglion, thus leading a basis for functional studies of these two distinct classes of neuropeptides. J. Comp. Neurol. 517:551,563, 2009. © 2009 Wiley-Liss, Inc. [source] Identification and Functional Characterization of the Delta 6-Fatty Acid Desaturase Gene from Thamnidium elegansTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 1 2007DEPEI WANG ABSTRACT. A cDNA sequence was cloned from the filamentous fungus Thamnidium elegans As3.2806 using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends method (RACE). Sequence analysis indicated that this cDNA sequence has an open reading frame of 1,380 bp, which encodes a 52.4 kDa peptide of 459 amino acids. The designated amino acid sequence has high similarity with that found in fungal delta 6-fatty acid desaturases: it shows three conserved histidine-rich motifs and two hydrophobic domains. A cytochrome b5-like domain was observed at the N-terminus. To elucidate the function of this novel putative desaturase, the open reading frame was cloned into the intracellular expression vector pPIC3.5K and the gene was expressed heterologously in Pichia pastoris. Accumulation of ,-linolenic acid to the level of 6.83% in total fatty acid demonstrated that the deduced amino acid sequence possesses of delta 6-fatty acid desaturase activity. [source] Wavelet analysis and the governing dynamics of a large-amplitude mesoscale gravity-wave event along the East Coast of the United StatesTHE QUARTERLY JOURNAL OF THE ROYAL METEOROLOGICAL SOCIETY, Issue 577 2001Fuqing Zhang Abstract Detailed diagnostic analyses are performed upon a mesoscale numerical simulation of a well-observed gravity-wave event that occurred on 4 January 1994 along the East Coast of the United States. The value of using wavelet analysis to investigate the evolving gravity-wave structure and of using potential vorticity (PV) inversion to study the nature of the flow imbalance in the wave generation region is demonstrated. The cross-stream Lagrangian Rossby number, the residual in the nonlinear balance equation, and the unbalanced geopotential-height field obtained from PV inversion are each evaluated for their usefulness in diagnosing the flow imbalance. All of these fields showed clear evidence of strong imbalance associated with a middle-to-upper tropospheric jet streak, and tropopause fold upstream of the large-amplitude gravity wave several hours before the wave became apparent at the surface. Analysis indicates that a train of gravity waves was continuously generated by geostrophic adjustment in the exit region of the unbalanced upper-level jet streak as it approached the inflection axis in the height field immediately downstream of the maximum imbalance associated with the tropopause fold. A split front in the middle troposphere, characterized by the advance of the dry conveyor belt above the warm front, was overtaken by one of these propagating waves. During this merger process, a resonant interaction resulted, which promoted the rapid amplification and scale contraction of both the incipient wave (nonlinear wave development) and the split front (frontogenesis). The gravity wave and front aloft became inseparable following this merger. The situation became even more complex within a few hours as the vertical motion enhanced by this front-wave interaction acted upon a saturated, potentially unstable layer to produce elevated moist convection. An analysis of the temporal changes in the vertical profile of wave energy flux suggests that moist convective downdraughts efficiently transported the wave energy from the midlevels downward beneath the warm-front surface, where the wave became ducted. However, pure ducting was not sufficient for maintaining and amplifying the waves; rather, wave-CISK (Conditional Instability of the Second Kind) was crucial. This complex sequence of nonlinear interactions produced a long-lived, large-amplitude gravity wave that created hazardous winter weather and disrupted society over a broad and highly populated area. Although gravity waves with similar appearance to this large-amplitude wave of depression occasionally have been seen in other strong cyclogenesis cases involving a jet streak ahead of the upper-level trough axis, it is unknown whether other such events share this same sequence of interactions. [source] Nucleotide polymorphisms and the 5,-UTR transcriptional analysis of the bovine growth hormone secretagogue receptor 1a (GHSR1a) geneANIMAL SCIENCE JOURNAL, Issue 5 2010Masanori KOMATSU ABSTRACT Growth hormone secretagogue receptor 1a (GHSR1a) mediates the different actions of its endogenous ligand, ghrelin. Ghrelin-GHSR is involved in many important functions that include growth hormone secretion and food intake. We evaluated the haplotype variety and characterized the microsatellite ((TG)n, 5,-UTR) and nucleotide polymorphisms of the bovine GHSR1a gene. The nucleotide sequencing of this gene (,6 kb) revealed 47 single nucleotide polymorphisms (SNPs), four indels and the microsatellite ((GTTT)n, Intron 1). The 19 haplotypes were constructed from all nucleotide viability patterns and were divided into three major groups. Four SNPs (L24V, nt456(G>A), D191N and nt667(C>T)) and DelR242 in Exon 1 and a haplotype block of approximately 2.2 kb (nt667(C>T) , nt2884 (A>G)) were found in Bos taurus breeds. Breed differences in allele frequencies of the two microsatellites, nt-7(C>A), L24V, and DelR242 loci were found (P < 0.005). A DelR242 was found in the Japanese Shorthorn (frequency: , 0.44), Japanese Brown, five European cattle breeds, the Philippine native cattle, but none detected in the Japanese Black or the Mishima island cattle. Additionally, 5,-rapid amplification of cDNA ends and RT-PCR analyses revealed that there were two different kinds of transcripts: spliced, without a microsatellite within 5,-UTR (GHSR1a); and non-spliced, with the microsatellite (GHSR1b). [source] Purification and expression of a protein elicitor from Alternaria tenuissima and elicitor-mediated defence responses in tobaccoANNALS OF APPLIED BIOLOGY, Issue 3 2010J. Mao A new protein elicitor, PeaT1, was purified from the mycelium of Alternaria tenuissima by column chromatography. PeaT1 was identified as a heat-stable and acidic protein. It induced systemic acquired resistance to tobacco mosaic virus (TMV) in tobacco plants but did not cause hypersensitive response. The elicitor-encoding gene was cloned by rapid amplification of cDNA ends method. Sequence analysis revealed that the cDNA is 624 bp in length and the open reading frame encodes for a polypeptide of 207 amino acids with a nascent polypeptide-associated complex domain. The peaT1 gene was cloned into the expression vector pET-28a and transformed into Escherichia coli BL21 (DE3). The recombinant elicitor also triggered defence responses in intact tobacco plants. The availability of the pure protein offers the possibility to isolate the corresponding receptor and links it to the downstream signalling pathway. [source] The responsive expression of heat shock protein 22 gene in zhikong scallop Chlamys farreri against a bacterial challengeAQUACULTURE RESEARCH, Issue 2 2010Lei Zhang Abstract HSP22 is a member of a small HSP subfamily contributing to the growth, transformation and apoptosis of the cell as well as acting as a molecular chaperone. In the present study, CfHSP22 cDNA was cloned from Chlamys farreri by the rapid amplification of cDNA ends technique. The full-length cDNA of CfHSP22 was of 1279 bp, consisting of a 5,-terminal untranslated region (5,UTR) of 122 bp, a 3,UTR of 581 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame of 576 bp encoding a polypeptide with a molecular mass of 22.21 kDa and a predicted isoelectric point of 9.69. There was an ,-crystallin domain, a hallmark of the sHSP subfamily, in the C-terminus, and the deduced amino acid sequence of CfHSP22 showed high similarity to previously identified HSP22s. CfHSP22 was constitutively expressed in the haemocyte, muscle, kidney, gonad, gill, heart and hepatopancreas, and the expression level in the hepatopancreas was higher than that in the other tissues. CfHSP22 transcription was up-regulated and reached a maximal level at 12 h after the bacterial challenge, and then declined progressively to the original level at 48 h. These results suggested that CfHSP22 perhaps play a critical role in response to the bacterial challenge in haemocytes of scallop C. farreri. [source] Cloning and expression analysis of a cDNA encoding lipoprotein lipase from the liver of adult grass carp (Ctenopharyngodon idella)AQUACULTURE RESEARCH, Issue 16 2009Han-Liang Cheng Abstract A full-length cDNA coding lipoprotein lipase (LPL) was cloned from the liver of adult grass carp (Ctenopharyngodon idella) using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends approaches. The cDNA obtained was 2414 bp long with a 1524 bp open reading frame encoding 507 amino acids, including a putative signal peptide 21 amino acids long. The LPL protein has a calculated molecular weight of 57.77 kDa and an isolectric point of 8.132. The main domains of LPL, such as catalytic site, disulphide bridge, N-linked glycosylation site, heparin-binding domain, lipid-binding site and site of dimer formation, are basically conserved between the grass carp and other vertebrates. The tissue distribution of LPL mRNA in the liver, head kidney, mesenteric adipose tissue, heart and white muscle of adult grass carp was analysed using the semi-quantitative RT-PCR method using ,-actin gene as an internal control; the result showed that the expressions of LPL mRNA were detected in all examined tissues of adult grass carp. The expression levels of LPL in the mesenteric adipose tissue were the highest among these tissues, followed by the liver and head kidney and the lowest expression was found in the heart and white muscle. [source] Molecular characterization of two nicotinic acetylcholine receptor subunits from Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae)ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 1 2009Pei-An Tang Abstract Two nicotinic acetylcholine receptor (nAChR) subunit genes, Lb,1 and Lb,8, were isolated and characterized from psocid, Liposcelis bostrychophila Badonnel, using the rapid amplification of cDNA ends (RACE) technique. They are the first two nAChR family members isolated from the insect order of Psocoptera. The full-length cDNAs of Lb,1 (GenBank accession number: EU871527) and Lb,8 (EU871526) consist of 2,025 and 1,763 nucleotides, respectively, and an open reading frame of 1,644 and 1,608,bp encoding 547 and 535 amino acid proteins, respectively. Both genes have typical features of nAChR family members, though they share only 56% identity in amino acid sequence. The dendrogram generated by the MEGA 3.1 program shows that the protein deduced by Lb,1 had the closest phylogenetic relationship to Agam,1 from Anopheles gambiae and Amel,1 from Apis mellifera, and Lb,8 shares the highest identity with Agam,8 from An. gambiae and Amel,8 from A. mellifera. Quantitative real-time PCR analysis showed that Lb,1 was expressed 2.03,6.54-fold higher than Lb,8 at the different developmental stages of L. bostrychophila. The highest expression levels of Lb,1 and Lb,8 were both detected at adult stage and the lowest were at the third and fourth nymphal stages, respectively. There was a stable and relatively low expression level for Lb,1, whereas there was a descending expression pattern for Lb,8 in the 1st through the 4th nymphal stadia. © 2009 Wiley Periodicals Inc. [source] Knockdown of Fc, receptor III in an arthritic temporomandibular joint reduces the nociceptive response in ratsARTHRITIS & RHEUMATISM, Issue 10 2010Phillip R. Kramer Objective Fc, receptor III (Fc,RIII; CD16) is a receptor expressed on immune cells that selectively binds IgG molecules. IgG binding results in cellular activation and cytokine release. IgG is an important factor in arthritis and can be found in the arthritic temporomandibular joint (TMJ). We undertook this study to test the hypothesis that a reduction in Fc,RIII expression in TMJ tissues would reduce the nociceptive and inflammatory responses in an inflamed joint. Methods Small interfering RNA (siRNA), either naked or complexed with linear polyethyleneimine, was injected into the superior joint space of the TMJ in rats. After administration of siRNA the joint was injected with saline or with Freund's complete adjuvant to induce arthritis. Nociceptive responses were quantitated in the rat by measuring the animal's meal duration. Fc,RIII expression in the TMJ tissue was assayed by immunocytochemistry or Western blotting. Cleavage of Fc,RIII transcript was then assayed by 5, rapid amplification of complementary DNA ends. Interleukin-1, (IL-1,) and IgG content was measured in the TMJ tissue by enzyme-linked immunosorbent assay. Results Injection of Fc,RIII siRNA reduced the amount of Fc,RIII in the TMJ tissues, and the transcript was cleaved in a manner consistent with an RNA interference mechanism. Moreover, injection of Fc,RIII siRNA reduced the nociceptive response of rats with an arthritic TMJ and reduced the amount of the proinflammatory cytokine IL-1,. Conclusion Fc,RIII contributes to the pain resulting from inflammatory arthritis of the TMJ, and siRNA has the potential to be an effective treatment for this disorder. [source] Fusion of PDGFRB to two distinct loci at 3p21 and a third at 12q13 in imatinib-responsive myeloproliferative neoplasmsBRITISH JOURNAL OF HAEMATOLOGY, Issue 2 2010Claire Hidalgo-Curtis Summary We identified four patients who presented with BCR-ABL1 negative myeloproliferative neoplasms and cytogenetically visible abnormalities of chromosome band 5q31-35. Fluorescence in situ hybridization indicated that the platelet-derived growth factor receptor , gene (PDGFRB) was disrupted in all four cases and 5, rapid amplification of cDNA ends identified in-frame mRNA fusions between PDGFRB and WDR48 (3p21), GOLGA4 (3p21) and BIN2 (12q13). Strikingly, all three genes encode proteins involving intracellular trafficking. Imatinib, a known inhibitor of PDGFR,, selectively blocked the growth of t(3;5) myeloid colonies and produced clinically significant responses in all patients. We conclude that PDGFRB fuses to diverse partner genes in atypical myeloproliferative neoplasms (MPNs). Although very rare, identification of these fusions is critical for proper management of affected individuals. [source] Molecular cloning and immunoglobulin E reactivity of a natural rubber latex lecithinase homologue, the major allergenic component of Hev b 4CLINICAL & EXPERIMENTAL ALLERGY, Issue 11 2005E. Sunderasan Summary Background Hev b 4 is an allergenic natural rubber latex (NRL) protein complex that is reactive in skin prick tests and in vitro immunoassays. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Hev b 4 is discerned predominantly at 53,55 kDa together with a 57 kDa minor component previously identified as a cyanogenic glucosidase. Of the 13 NRL allergens recognized by the International Union of Immunological Societies, the 53,55 kDa Hev b 4 major protein is the only candidate that lacks complete cDNA and protein sequence information. Objective We sought to clone the transcript encoding the Hev b 4 major protein, and characterize the native protein and its recombinant form in relation to IgE binding. Methods The 5,/3, rapid amplification of cDNA ends method was employed to obtain the complete cDNA of the Hev b 4 major protein. A recombinant form of the protein was over-expressed in Escherichia coli. The native Hev b 4 major protein was deglycosylated by trifluoromethane sulphonic acid. Western immunoblots of the native, deglycosylated and recombinant proteins were performed using both polyclonal antibodies and sera from latex-allergic patients. Results The cDNA encoding the Hev b 4 major protein was cloned. Its open reading frame matched lecithinases in the conserved domain database and contained 10 predicted glycosylation sites. Detection of glycans on the Hev b 4 lecithinase homologue confirmed it to be a glycoprotein. The deglycosylated lecithinase homologue was discerned at 40 kDa on SDS-PAGE, this being comparable to the 38.53 kDa mass predicted by its cDNA. Deglycosylation of the lecithinase homologue resulted in the loss of IgE recognition, although reactivity to polyclonal rabbit anti-Hev b 4 was retained. IgE from latex-allergic patients also failed to recognize the non-glycosylated E. coli recombinant lecithinase homologue. Conclusion The IgE epitopes of the Hev b 4 lecithinase homologue reside mainly in its carbohydrate moiety, which also account for the discrepancy between the observed molecular weight of the protein and the value calculated from its cDNA. [source] | ||||||||||||||||||||||||||||