1000 resultados para Insects Identification


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Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a crucial component of almost the entire tumor necrosis factor receptor superfamily signaling pathway. In the present study, a TRAF2 gene has been cloned from grass carp (Ctenopharyngodon idella) by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The full-length cDNA is 3162 bp, including a 60 bp 5' untranslated region (UTR), a 1611 bp open reading frame, and a 1491 bp 3' UTR. The polyadenylation signal (AATAAA) and the mRNA instability motifs (ATTTTA, ATTTA) were followed by a poly(A) tail in the 3' UTR. No signal peptide or transmembrane region has been found in the putative amino acids of grass carp TRAF2 (gcTRAF2). Phylogenetic tree analysis clearly showed that gcTRAF2 is nearest to the TRAF2 gene of goldfish. The identity of gcTRAF2 with its homologs in other vertebrates ranges from 56% to 97%. It is characterized by one RING-type signature at the N-terminus, one zinc finger in the middle part, and one conserved TRAF domain consisting of a C-proximal (TRAF-C) subdomain and a N-proximal (TRAF-N) subdomain. The identity of TRAF-C among all TRAF2 homologs in vertebrates varies from 78% to 97%, whereas the identity of TRAF-N ranges from 56% to 100%. The recombinant gcTRAF2 has been expressed in Escherichia coli using pET-32a expression vector. The rabbit anti-gcTRAF2 polyclonal antibody was obtained. The expression of gcTRAF2 in different organs was examined by real-time quantitative polymerase chain reaction and Western blot analysis. It was widely distributed in heart, head kidney, thymus, brain, gill, liver, spleen, and trunk kidney. This is the first report of a TRAF2 homolog molecule in fish.

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Cross-species nuclear transfer (NT) has been used to retain the genetic viability of a species near extinction. However, unlike intra-species NT, most embryos produced by cross-species NT were unable to develop to later stages due to incompatible nucleocytoplasmic interactions between the donor nuclei and the recipient cytoplasm from different species. To study the early nucleocytoplasmic interaction in cross-species NT, two laboratory fish species (zebrafish and rare minnow) from different subfamilies were used to generate cross-subfamily NT embryos in the present study. Suppression subtractive hybridization (SSH) was performed to screen out differentially expressed genes from the forward and reverse subtractive cDNA libraries. After dot blot and real-time PCR analysis, 80 of 500 randomly selective sequences were proven to be differentially expressed in the cloned embryos. Among them, 45 sequences shared high homology with 28 zebrafish known genes, and 35 sequences were corresponding to 22 novel expressed sequence tags (ESTs). Based on functional clustering and literature mining analysis, up-and down-regulated genes in the cross-subfamily cloned embryos were mostly relevant to transcription and translation initiation, cell cycle regulation, protein binding, etc. To our knowledge, this is the first report on the determination of genes involved in the early development of cross-species NT embryos of fish. (C) 2007 Elsevier Inc. All rights reserved.

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In this study, an alternative splicing transcript GtH-alpha 291 was identified by RT-PCR, which is 291 nt and exists not only in the pituitary but also in the ovary in common carp Cyprinus carpio. The analysis of GtH-alpha 291 amino acid sequence by the SignalP server predicted that the 'missing segment' might characterize as a signal peptide. In the secretion experiment, GtH-alpha 357 subunit could be secreted out of HeLa cells while GtH-alpha 291 could not, which confirmed the prediction. Co-immunoprecipitation assay proved that GtH-alpha 291 subunit is able to interact with both FSH-beta and LH-beta as GtH-alpha 357 does. This is the first report concerning an alternative splicing transcript of a GtH alpha subunit. Further studies are necessary to elucidate the specific role of this variant in the regulation of gonadal development and sexual maturation. (c) 2007 The Authors.

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Myelin basic protein (MBP), as a major component of the myelin sheath, has been revealed to play an important role informing and maintaining myelin structure in vertebrate nervous system. In teleost, hypothalamus is an instinctive brain center and plays significant roles in many physiological functions, such as energy metabolism, growth, reproduction, and stress response. In comparison with other MBP identified in vertebrates, a smallest MBP is cloned and identified from the orange-spotted grouper hypothalamic cDNA plasmid library in this study. RT-PCR analysis and Western blot detection indicate that the EcMBP is specific to hypothalamus, and expresses mainly in the tuberal hypothalamus in adult grouper. Immunofluorescence localization suggests that EcMBP should be expressed by oligodendrocytes, and the expressing cells should be concentrated in hypothalamus and the area surrounding hypothalamus, such as NPOpc, VC, DP, NLTm, and NDLI The studies on EcMBP expression pattern and developmental behaviour in the brains of grouper embryos and larvae reveal that the EcMBP-expressing cells are only limited in a defined set of cells on the border of hypothalamus, and suggest that the EcMBP-expressing cells might be a subpopulation of oliaodendrocyte progenitor cells. This study not only identifies a smallest MBP isoform specific to hypothalamus that can be used as a molecular marker of oligodendrocytes in fish, but also provides new insights for MBP evolution and cellular distribution. (C) 2007 Elsevier B.V.. All rights reserved.

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Blood smears and purified trypanosome from freshwater fishes yellow catfish (Pseudobagras fulvidraco) and common carp (Cyprinus carpio) captured from Niushan Lake, Hubei Province were examined to determine whether all of their trypanosomes were Trypanosoma pseudobagri, a species of supposed host specificity and widespread existence across China. Trypanosomes occurred in 16/16 blood smears, and morphometric character analysis of trypanosomes from these smears showed that there were three morphospecies, Trypanosoma sp Carpio, T. sp Pseudobagri, and T. sp. 18S rDNA sequences of trypanosomes from 16 samples revealed three genetic groups among these fish trypanosomes. Group 1 was from C. carpio containing T. sp Carpio; groups 2 and 3 were from P. fulvidraco containing T. sp Pseudobagri and T. sp, respectively. The high similarity of morphometric characters and 18S rDNA sequences showed that T. sp Carpio and T. siniperca probably were the same species. T. sp Pseudobagri was the first occurrence in China. Sequence comparison showed that T. sp Pseudobagri sequence was most similar to that of clone Marv, whereas T. sp sequence differ from those of T. sp Carpio and T. sp Pseudobagri by 5.4 and 5.8%, respectively, and tentatively identified as T. pseudobagri. It was concluded that three species of trypanosomes, at least three genotypes occur in Niushan Lake fishes, and P. fulvidraco in this region appear to contain both types, although the identification of T. pseudobagri remains a problem.

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ISG15 is one of the most strongly induced genes upon viral infection, interferon (IFN) stimulation, and lipopolysaccharide, (LPS) stimulation, and only one copy has been found in mammals so far. Here two fish ISG15 genes, termed CaISG15-1 and CaISG15-2, have been cloned and sequenced from UV-inactivated GCHV (grass carp haemorrhagic virus)-infected and IFN-produced CAB cells (crucian carp Carassius auratus blastulae embryonic cells) by suppression subtractive hybridization. The full-length cDNA sequences of two crucian carp ISG15 encode a 155-amino-acid protein and a 161-amino-acid protein, both of which show 78.9% identity overall and possess the characteristic structures of mammalian ISG15 proteins including two tandem ubiquitin-like domains and the C-terminal canonical LRLRGG motif. In CAB cells treated with different stimuli including active virus, UV-inactivated GCHV and IFN containing supernatant (ICS), the expression of both CaISG15-1 and CaISG15-2 was up-regulated but displayed different kinetics. Poly I:C and LPS were also able to induce an increase in mRNA for both genes. In CAB cells responsive to active GCHV, UV-inactivated GCHV, CAB ICS, Poly 1:12 and LPS, CaISG15-1 was upregulated more significantly than CaISG15-2. These results suggest that there are two ISG15 homologues in crucian carp, both of which might play distinct roles in innate immunity against viral and bacterial infection. (c) 2006 Elsevier Ltd. All rights reserved.

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Synechocystis sp. PCC 6803 exposed to chill (5 degrees C)-light (100 mu mol photons m(-2) s(-1)) stress loses its ability to reinitiate growth. From a random insertion mutant library of Synechocystis sp. PCC 6803, a sll1242 mutant showing increased sensitivity to chill plus light was isolated. Mutant reconstruction and complementation with the wild-type gene confirmed the role of sll1242 in maintaining chill-light tolerance. At 15 degrees C, the autotrophic and mixotrophic growth of the mutant were both inhibited, paralleled by decreased photosynthetic activity. The expression of sll1242 was upregulated in Synechocystis sp. PCC 6803 after transfer from 30 to 15 degrees C at a photosynthetic photon flux density of 30 mu mol photons m(-2) S-1. sll1242, named ccr (cyanobacterial cold resistance gene)- 1, may be required for cold acclimation of cyanobacteria in light.

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Angel-related element belongs to the family of miniature inverted-repeat transposable elements (MITEs). In this paper we report the identification of an Angel-related element in the series Leuciscini of cyprinid fishes, which is located in the second intron of the growth hormone (GH) gene. We have also found that this element is absent in orthologous locus in the series Barbini of cyprinid fishes, that provides new evidence for the monophyly of the series Leuciscini. The insertion of Angel-related element into the GH gene might take place in the common ancestor of the series Leuciscini after its divergence from the series Barbini. The high sequence divergence and relatively broad species distribution of Angel-related elements implies that they might be ancient transposons which appeared about 26 million years ago.

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The unusual allotetraploid form with unequal contribution of chromosome sets was discovered from the gynogenetic offspring of Carassius auratus gibelio stimulated by red common carp sperm. In this study, genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) with 45S rDNA probe are used. The GISH results lead to the identification of species-specific chromosomes, which permits to demonstrate the origin and genome organization in the allotetraploid form. Moreover, chromosome localization of 45S rDNA and co-localizations of 45S rDNA and Cyprinus carpio genomic DNA further confirm that one extra 45S rDNA positive chromosome in the allotetraploid form originates from the paternal haploid genome of C carpio, and other 5 45S rDNA-containing chromosomes are from the maternal genome of Carassius auratus gibelio. And, the correlation between 45 rDNA and the nucleolar organizer regions (NORs) is confirmed by silver nitrate staining. The data provide direct experiment evidence that the allotetraploid actually contains three chromosome sets of Carassius auratus gibelio and one chromosome set of C carpio, and will be a useful genetic material for both basic research and breeding practice. (c) 2006 Elsevier B.V. All rights reserved.

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Short interspersed nuclear elements (SINEs) are widespread among eukaryotic genomes. They are repetitive DNA sequences that have been amplified by retrotransposition. In this study, a class of SINEs were isolated from the Opsariichthys bidens genome, and named Opsar. Sequence analysis confirmed that Opsar is a new class of typical SINEs derived from tRNA molecules. With the tRNA-derived region of Opsar and through BLASTN search, we further identified Zb-SINEs from the zebrafish genome, which includes two groups: Zb-SINE-A and Zb-SINE-B. The Zb-SINE-A group comprises subfamilies of -Al--A5, and the Zb-SINE-B group is a dimer of the tRNA(Ala)-derived region and shares a similar dimeric composition to Alu. Zb-SINEs are composed of three distinct regions: a 5 end tRNA-derived region, a tRNA-unrelated region and a 3 end AT-rich region. The flanking regions are AT rich. The average length of Zb-SINEs elements is about 340 6p. Zb-SINEs account for as much as 0.1% of the whole zebrafish genome. About 70% of the Zb-SINEs are on chromosomes 11, 18, and 19. These Zb-SINEs were characterized by PCR and dot hybridization. The distribution pattern of Zb-SINEs in genome strongly supports the master genes model. The tRNA-derived regions of Opsar and Zb-SINEs were compared with the tRNA(Ala) gene, and they showed 76% similarity, indicating that Opsar and Zb-SINEs originated from an inactive tRNA(Ala) sequence or a tRNA(Ala)-like sequence. In view of the evolutionary status of zebrafish in the Cyprinidae, we deduced that Zb-SINEs were a very old class of interspersed sequences.

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Previous studies have demonstrated that germinal vesicle of amphibian oocyte contains small nuclear ribonucleoprotein polypeptide C (SNRPC). In this study, a putative member of SNRPC was identified from Carassius auratus gibelio oocyte cDNA library. Its full-length cDNA has an open reading frame of 201 nt for encoding a peptide of 66 an, a short 5'-UTR of 19 nt and a long 3'-UTR of 347 nt including a polyadenylation signal and poly- (A) tail, and the deduced amino acid sequence has 47% identity with the C-terminal of the zebrafish small nuclear ribonucleoprotein polypeptide C. Western blot analysis revealed its oocyte-specific expression. Immunofluorescence localization indicated that its gene product localized to numerous nucleoli within the oocytes and showed dynamic changes with the nucleoli during oocyte maturation. RT-PCR and Western blot analysis further revealed its constant presence in the oocytes and in the embryos until hatching. The data suggested that the newly identified CagOSNRPC might be a nucleolar protein. (c) 2006 Elsevier Inc. All rights reserved.

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The cDNA of growth hormone receptor (GHR) was cloned from the liver of 2-year common carp (Cyprinus carpio L.) by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE). Its open reading frame (ORF) of 1806 nucleotides is translated into a putative peptide of 602 amino acids, including an extracellular ligand-binding domain of 244 amino acids (aa), a single transmembrane domain of 24 aa and an intracellular signal-transduction domain of 334 aa. Sequence analysis indicated that common carp GHR is highly homologous to goldfish (Carassius auratus) GHR at both gene and protein levels. Using a pair of gene-specific primers, a GHR fragment was amplified from the cDNA of 2-year common carp, a 224 bp product was identified in liver and a 321 bp product in other tissues. The sequencing of the products and the partial genomic DNA indicated that the difference in product size was the result of a 97 bp intron that alternatively spliced. In addition, the 321 bp fragment could be amplified from all the tissues of 4-month common carp including liver, demonstrating the occurrence of the alternative splicing of this intron during the development of common carp. Moreover, a semi-quantitative RT-PCR was performed to analyze the expression level of GHR in tissues of 2-year common carp and 4-month common carp. The result revealed that in the tissues of gill, thymus and brain, the expression level of GHR in 2-year common carp was significantly tower than that of 4-month common carp.

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White spot syndrome virus (WSSV) is one of the most significant viral pathogens causing high mortality and economic damage in shrimp aquaculture. Although intensive efforts were undertaken to detect and characterize WSSV infection in shrimp during the last decade, we still lack methods either to prevent or cure white spot disease. Most of the studies on neutralizing antibodies from sera have been performed using in vivo assays. For the first time, we report use of an in vitro screening method to obtain a neutralizing scFv antibody against WSSV from a previously constructed anti-WSSV single chain fragment variable region (scFv) antibody phage display library. From clones that were positive for WSSV by ELISA, 1 neutralizing scFv antibody was identified using an in vitro screening method based on shrimp primary lymphoid cell cultures. The availability of a neutralizing antibody against the virus should accelerate identification of infection-related genes and the host cell receptor, and may also enable new approaches to the prevention and cure of white spot disease.

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Taenia solium metacestode, a larval pork tapeworm, is a causative agent of neurocysticercosis, one of the most common parasitic diseases in the human central nervous system. In this study, we identified a cDNA encoding for a cathepsin L-like cysteine protease from the T solium metacestode (TsCL-1) and characterized the biochemical properties of the recombinant enzyme. The cloned cDNA of 1216 bp encoded 339 amino acids with an approximate molecular weight of 37.6 kDa which containing a typical signal peptide sequence (17 amino acids), a pro-domain (106 amino acids), and a mature domain (216 amino acids). Sequence alignments of TsCL-1 showed low sequence similarity of 27.3-44.6 to cathepsin L-like cysteine proteases from other helminth parasites, but the similarity was increased to 35.9-55.0 when compared to mature domains. The bacterially expressed recombinant protein (rTsCL-1) did not show enzyme activity; however, the rTsCL-1 expressed in Pichia pastoris showed typical biochemical characteristics of cysteine proteases. It degraded human immunoglobulin G (IgG) and bovine serum albumin (BSA), but not collagen. Western blot analysis of the rTsCL-1 showed antigenicity against the sera from patients with cysticercosis, sparganosis or fascioliasis, but weak or no antigenicity against the sera from patients with paragonimiasis or clonorchiasis. (c) 2006 Published by Elsevier B.V.

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In a previous study, a scFv phage display library against white spot syndrome virus (WSSV) was constructed and yielded a clone designated A I with conformational specificity against native but not denatured viral antigen. Although the clone A1 has been used successfully as a diagnostic antibody, its precise target antigen has not been elucidated. A different strategy was adopted involving the construction of a second T7 phage display library utilizing mRNA isolated from shrimp infected with WSSV. Following RT-PCR and T7 phage library construction, phages displaying the candidate epitope were selected with A I scFv. Since successive enrichment steps were not associated with an increased titer of the phages, enrichment after successive tests was confirmed by PCR resulting in the prefer-red selection of a specific DNA sequence encoding a novel nucleocapsid protein WSSV388. Immune electron microscopy revealed that WSSV388 is located on the nucleocapsid. This result demonstrated that unknown antigen could be identified by phage display using the epitope conformation dependent scFv. (c) 2006 Elsevier B.V. All rights reserved.