Molecular cloning, promoter analysis and induced expression of the complement component C9 gene in the grass carp Ctenopharyngodon idella

Complement-mediated killing of pathogens through lytic pathway is an important effector mechanism of innate immune response. C9 is the ninth member of complement components, creating the membrane attack complex (MAC). In the present study, a putative cDNA sequence encoding the 650 amino acids of C9 and its genomic organization were identified in grass carp Ctenopharyngodon idella. The deduced amino acid sequence of grass carp C9 (gcC9) showed 48% and 38.5% identity to Japanese flounder and human C9, respectively. Domain search revealed that gcC9 contains a LDL receptor domain, an EGF precursor domain, a MACPF domain and two TSP domain located in the N-terminal and C-terminal, respectively. Phylogenetic analysis demonstrated that gcC9 is clustered in a same clade with Japanese flounder, pufferfish and rainbow trout C9. The gcC9 gene consists of 11 exons with 10 introns, spacing over approximately 7 kb of genomic sequence. Analysis of gcC9 promoter region revealed the presence of a TATA box and some putative transcription factor such as C/EBP, HSF, NF-AT, CHOP-C, HNF-3B, GATA-2, IK-2, EVI- 1, AP-1, CP2 and OCT-1 binding sites. The first intron region contains C/EBPb, HFH-1 and Oct-1 binding sites. RT-PCR and Western blotting analysis demonstrated that the mRNA and protein of gcC9 gene have similar expression patterns, being constitutively expressed in all organs examined of healthy fish, with the highest level in hepatopancreas. By real-time quantitative RT-PCR analysis, gcC9 transcripts were significantly up-regulated in head kidney, spleen, hepatopancreas and down-regulated in intestine from inactivated fish bacterial pathogen Flavobacterium columnare-stimulated fish, demonstrating the role of C9 in immune response. (c) 2007 Elsevier B.V. All rights reserved.

Gene structure and transcription of IRF-1 and IRF-7 in the mandarin fish Siniperca chuatsi

The genes of IRF-1 and IRF-7 have been cloned from the mandarin fish (Siniperca chuatsi). The IRF-1 gene has 4919 nucleotides (nt) and contains 10exons and 9introns, with an open reading frame (ORF) of 903 ntencoding301 aa. The IRF-7 gene has 6057 nt and also contains 10exons and 9introns, with an ORF of 1308 nt encoding 436 aa. The IRF-1 and IRF-7 genes have only one copy each in the genome. The transcription of IRF-1 and IRF-7 in different organs was analyzed by real-time PCR, and both molecules were constitutively expressed. The IRF-I and IRF-7 mRNAs were abundant in gill, spleen, kidney and pronephros. The temporal transcriptional changes for IRF-1, IRF-7 and Mx were investigated within 48 h after poly I: C stimulation in liver, gill, spleen and pronephros. An increased transcription was detected for IRF-1 and IRF-7 12 h post-stimulation, being earlier than the transcription of Mx protein; however, IRF-1 and IRF-7 transcription decreased while the Mx protein was stable at 48 h post-stimulation. (c) 2007 Published by Elsevier B.V.

The acute effects of microcystin LR on the transcription of nine glutathione S-transferase genes in common carp Cyprinus carpio L.

The glutathione S-transferases play important roles in the detoxification of microcystin. In this experiment, nine glutathione S-transferase genes including cytosolic GSTs (rho, mu, theta, alpha and pi), mitochondrial GST (kappa) and microsomal GSTs (mGST1, mGST2 and mGST3) were cloned from common carp Cyprinus carpio. The mRNA abundance of each carp GST isoform in liver was analyzed by real time PCR. The relative changes after stimulation with microcystin LR were also analyzed: increased levels of transcription of GST alpha, rho and mGST3 isoforms were detected at 6 h post stimulation; the transcription of mu, theta and mGST2 isoforms were relatively stable; and all the GST isoforms except GST kappa and rho recovered to original levels compared with controls at 72 h. It is suggested that MC-LR showed different effects on the transcription of nine carp GST isoforms. (c) 2006 Elsevier B.V. All rights reserved.

Gene structure and transcription of IRF-2 in the mandarin fish Siniperca chuatsi with the finding of alternative transcripts and microsatellite in the coding region

The gene of interferon regulatory factor-2 (IRF-2) has been cloned from the mandarin fish (Siniperca chuatsi). The IRF-2 gene has 6,418 nucleotides (nt) and contains eight exons and seven introns, encoding two mRNAs. The two IRF-2 mRNAs each contained an open reading frame of 873 nt, which both translate into the same 291 amino acids but differed in their 5' untranslated region: one mRNA was transcribed initially from the exon 1 bypassing exon 2, while the other was transcribed from the exon 2. The microsatellites (CA repeats) could be found in the carboxyl terminal region of mandarin fish IRF-2, which result in the truncated form molecules. The microsatellites' polymorphism was investigated, and eight alleles were found in 16 individuals. The microsatellites were also examined in IRF-2 of several freshwater perciform fishes. The transcription of the IRF-2 in different tissues with or without poly inosine-cytidine stimulation was analyzed by real-time PCR, and the constitutive transcription of both molecules could be detected in all the tissues examined.

Agrobacterium-mediated transformation of Kentucky bluegrass

Embryogenic calli of Kentucky bluegrass, named Md, were induced from mature seeds and embryos, and proliferated on medium K3 containing 2,4-dichlorophenoxyacetic acid (2,4-D, 10.0 mumol/L), 6-benzylaminopurine (BAR, 0.5 mumol/L) and K5 which was the K3 medium supplemented with cupric sulfa (0.5 mumol/L) under dim-light condition (20-30 mumol.m(-2).s-1, 16 h light) at 24 degreesC. Embryogenic calli were transformed with plasmids pDM805 Carring bar and gus genes, Which was mediated by an Agrobacterium strain AGL1, four transgenic lines were obtained. The important factors that affect the transformation efficiency and obtain desirable number of transgenic plants included: (1) the quality of embryogenic calli; (2) light condition and time of co-cultivation; (3) concentration of antibiotics used for suppressing the overgrowth of Agrobacterium in the course of transformed plant regeneration; (4) selection pressure, etc. The micro nutrient of cupric had significant influence on the quality of embryogenic calli. This presentation is the first successful protocol of Kentucky bluegrass transformation mediated by Agrobacterium.

RecA-mediated, targeted mutagenesis in zebrafish

We have evaluated the efficacy of RecA, a prokaryotic protein involved with homologous recombination, to direct site-specific mutagenesis in zebrafish embryos. For this we coinjected a vector containing a mutated enhanced green fluorescent protein (EGFP) gene plus 236-nucleotide corrective single-stranded DNAs coated with RecA into I-cell zebrafish embryos. Twenty-hours after fertilization, about 5% to 20% of injected embryos showed EGFP expression in I or more cells when RecA-coated corrective DNAs were used, but not when RecA was omitted. Mutated EGFP genes with 1-bp insertions or deletions were inefficiently activated, whereas those with 7-bp insertions were activated about 4-fold more efficiently. RecA-coated template strand had a higher efficiency than its complementary strand in activation of EGFP expression. Prior irradiation of the embryos with UV light enhanced RecA-mediated restoration of gene activity, suggesting that the effects we observed were augmented by one or more factors of zebrafish DNA repair systems.

The onset of foreign gene transcription in nuclear-transferred embryos of fish

The transcriptional onset of hGH-transgene in fish was studied in the following three cases: the first is in MThGH-transgenic F-4 common carp (Cyprinus carpio) embryos, the second is in nuclear-transferred embryos supported by the transgenic F-4 embryonic nuclei, and the third is in nuclear-transferred embryos supported by the transgenic F-4 tail-fin nuclei. RT-PCR results show that the hGH-transgene initiates its transcriptional activity from early-gastrula stage, the early blastula stage and even 16-cell stage in the first, second and third cases, respectively. it looks like that fish egg cytoplasm could just offer a very restricted reprogramming on transcriptional activity of specific gene in differentiated cell nuclei by nuclear transplantation.

A detection method for grass carp hemorrhagic virus (GCHV) based on a reverse transcription polymerase chain reaction

A rapid, sensitive and highly specific detection method for grass carp hemorrhagic virus (GCHV) based on a reverse transcription-polymerase chain reaction (RT-PCR) has been developed. Two pairs of PCR primers were synthesized according to the cloned cDNA sequences of the GCHV-861 strain. For each primer combination, only one specific major product was obtained when amplification was performed by using the genomic dsRNA of GCHV-861 strain. The lengths of their expected products were 320 and 223 bp, respectively. No products were obtained when nucleic acids other than GCHV-861 genomic RNA were used as RT-PCR templates. To assess the sensitivity of the method, dilutions of purified GCHV-861 dsRNA total genome (0.01 pg up to 1000 pg) were amplified and quantities of as little as 0.1 pg of purified dsRNA were detectable when the amplification product was analyzed by 1.5% agarose gel electrophoresis. This technique could detect GCHV-861 not only in infected cell culture fluids, but also in infected grass carp Ctenopharyngodon idellus and rare minnow Gobiocypris rarus with or without hemorrhagic symptoms. The results show that the RT-PCR amplification method is useful for the direct detection of GCHV.

Hole-mediated electric field tunable high Curie temperature in Mn-doped wurtzite ZnO nanowires

The hole-mediated Curie temperature in Mn-doped wurtzite ZnO nanowires is investigated using the k center dot p method and mean field model. The Curie temperature T-C as a function of the hole density has many peaks for small Mn concentration (x(eff)) due to the density of states of one-dimensional quantum wires. The peaks of T-C are merged by the carriers' thermal distribution when x(eff) is large. High Curie temperature T-C > 400 K is found in (Zn,Mn)O nanowires. A transverse electric field changes the Curie temperature a lot. (Zn,Mn)O nanowires can be tuned from ferromagnetic to paramagnetic by a transverse electric field at room temperature. (c) 2007 American Institute of Physics.

Growth of Ge quantum dot mediated by boron on Ge wetting layer

We, report on the influence of boron on the formation of Ge quantum dots. The investigated structure consists of a Ge wetting layer, on which a sub-monolayer boron is deposited and subsequently a Ge top layer. For sufficiently thin Ge top layers, the strain field induced by boron on Ge wetting layer destabilizes the Ge top layer and causes the formation of small Ge quantum dots. However, for thicker Ge top layers, boron on the Ge wetting layer diffuses into Ge layers, compensates partly the strain and delays the evolution of Ge quantum dots. By this method, small Ge quantum dots with high density as well as size uniformity can be formed by optimizing the growth condition. (c) 2005 Elsevier B.V. All rights reserved.

Carrier channels of multimodal-sized quantum dots: A surface-mediated adatom migration picture

This paper focuses on the study of carrier channels of multimodal-sized quantum dots formed on patterned substrate by a rate-equation-based model. Surface-mediated indium adatom migration is revealed by a direct comparison between quantum dot wetting layer, which acts as carrier channel, formed on a flat substrate and on a patterned substrate. For the assessment of suitability, the carrier channel of the dot-in-well structure has also been studied by the present model, and the transition energies of the carrier channel (e.g., InGaAs quantum well) obtained from theoretical simulation agree fairly well with those obtained from the reflectance measurements.

Mechanism on exciton-mediated energy transfer in erbium-doped silicon

Exciton-mediated energy transfer model in Er-doped silicon was presented. The emission intensity is related to optically active Er concentration, lifetime of excited Er3+ ion and spontaneous emission. The thermal quenching of the Er luminescence in Si is caused by thermal ionization of Er-bound exciton complex and nonradiative energy back-transfer processes, which correspond to the activation energy of 6.6 and 47.4 meV, respectively. Er doping in silicon introduces donor states, a large enhancement in the electrical activation of Er (up to two orders of magnitude) is obtained by co-implanting Er with O. It appears that the donor states are the gateway to the optically active Er. (C) 2000 Elsevier Science B.V. All rights reserved.