339 resultados para cDNA membrane


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Natural resistance associated macrophage protein (Nramp) controls partially innate resistance to intracellular parasites. Its function is to enhance the ability of macrophages to kill pathogens. However, little is known about the structure and function of Nramp in lower vertebrates such as teleosts. We have recently isolated a cDNA encoding Nramp from Japanese flounder (Paratichthys olivaceus). The full-length cDNA of the Nramp is 3066 bp in length, including 224 bp 5' terminal UTR, 1662 bp encoding region and 1180 bp 3' terminal UTR. The 1662-nt open reading frame was found to code for a protein with 554 amino acid residues. Comparison of amino acid sequence indicated that Japanese flounder Nramp consists of 12 transmembrane (TM) domains. A consensus transport motif (CTM) containing 20 residues was observed between transmembrane domains 8 and 9. The deduced amino acid sequence of Japanese flounder had 77.30%, 82.71%, 82.67%, 79.64%, 80.72%, 90.97%, 91.16%, 60.14%, 71.48%, 61.69%, 72.37% identity with that of rainbow trout Nramp alpha and beta, channel catfish Nramp, fathead minnow Nramp, common carp Nramp, striped sea bass Nramp, red sea bream Nramp, mouse Nramp 1 and 2, human Nramp 1 and 2, respectively. RT-PCR indicated that Nramp transcripts were highly abundant in spleen, head kidney, abundant in intestine, liver and gill, and less abundant in heart. The level of Nramp mRNA in embryos gradually increases during embryogenesis from 4 h (8 cell stage) to 80 h (hatched stage) after fertilization. (c) 2005 Elsevier Ltd. All rights reserved.

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In order to identify genes encoding the outer membrane proteins (OMPs) of the myxobacter Flavobacterium columnare G(4), the expression library of the bacterium was screened by using rabbit antisera developed against its OMPs. Positive colonies of Escherichia coli M15 containing fragments encoding the bacterial OMPs were selected for cloning the relevant genes by genomic walking methods. Two genes encoding a membrane-associated zinc metalloprotease and prolyl oligopeptidase are reported in this paper. The membrane-associated zinc metalloprotease gene (map) is 1800 bp in length, coding for 449 amino acids (aa). Despite the presence of a conserved motif HEXXH for all metalloproteases, the special HEXXH similar to 32 aa similar to E motif of the F. columnare G(4) Map and its low level of identity with other reported zinc-containing metalloproteases may imply that the membrane-associated zinc metalloprotease of F. columnare G(4) represents a new family of zincins. The gene encoding prolyl oligopeptidase (Pop), a serine proteinase, is 2352 bp in length, coding for 649 aa. Sequence homology analysis revealed that the Pop is also novel as it has <50% identity with other reported prolyl oligopeptidase family proteins. The present study represents the first to employ anti-fish bacterial OMP sera to screen genes of membrane-associated proteases of fish pathogenic bacteria, and to provide necessary information for the examination of the role of the two genes in the infection and pathogenesis of F. columnare.

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Potential roles of Clq/tumor necrosis factor (TNF) superfamily proteins have been observed in vertebrate oogenesis and oocyte maturation, but no ovary-specific member has been identified so far. In this study, we have cloned and identified a novel member of Clq family with a Clq domain in the C-terminal from fully grown oocyte cDNA library of color crucian carp and demonstrated that the gene might be specifically expressed in ovary and therefore designated as Carassius auratus ovary-specific Clq-like factor, CaOClq-like factor. It encodes a 213 amino acid protein with a 17 amino acid signal peptide. There is only one protein band of about 24.5 kDa in the extracts from phase I to phase IV oocytes, but two positive protein bands are detected in the extracts of mature eggs and fertilized eggs. Furthermore, the mobility shift of the smaller target protein band cannot be eliminated by phosphatase treatment, but the larger protein band increases its mobility on the gel after phosphatase treatment, suggesting that the larger protein might be a phosphorylated form. Immunofluorescence localization indicates that the CaOClq-like proteins localize in cytoplasm, cytoplasm membrane and egg envelope of the oocytes at cortical granule stage and vitellogenesis stage, whereas they were compressed to cytoplasm margin in ovulated mature eggs and discharged into perivitelline space between cytoplasm membrane and egg envelope after egg fertilization. Further studies on distribution and translocation mechanism of the CaOClq-like factor will be benefit to elucidate the unique function in oogenesis, oocyte maturation and egg fertilization. (C) 2004 Elsevier Inc. All rights reserved.

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To understand the molecular events governing fish oogenesis, a multiple technique was used to identify the genes differentially expressed at different phases during fish oogenesis. This technique is a combination of suppression subtractive hybridization, SMART cDNA synthesis and RACE-PCR. Here we report the cDNA cloning and expression characterization of a novel SNX gene based on its differential transcription between previtellogenic and fully mature oocytes in naturally gynogenetic gibel carp. First, a cDNA fragment selectively expressed in previtellogenic oocytes was identified and used to screen a SMART cDNA library prepared from the same mRNA sample by RACE-PCR for cloning fully length cDNA. The full length cDNA was 1392-bp long and coded for a novel SNX protein with 225 amino acids. The 5' UTR had 72 bp and 3' UTR had 642 bp. Unlike most of maternal genes that are transcribed after vitellogenesis and stored in oocytes, this gene is expressed at a higher level in the previtellogenic oocytes and at a much lower level in fully matured oocytes. However, RT-PCR analysis of tissues showed it was ubiquitous transcription. The novel gene is named fish sorting nexin (fSNX), because it contains a conserved PX domain. The fact which major expression of the gene occurs in the previtellogenic oocytes suggests that it might have an important function in the oogenesis. (C) 2003 Elsevier Inc. All rights reserved.

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Immunoglobulin light chain cDNA sequences of a perciform fish, the mandarin fish Siniperca chuatsi were amplified from head kidney mRNA by reverse transcription (RT)-PCR and RACE methods using degenerated primer and gene specific ones. In cDNA sequences of the VL region, nucleotide exchanges were present mainly within CDRs, although a lesser degree of variability was also found in FRs. Moreover, the length of CDRI and CDR3 in the mandarin fish is shorter than in most other fish species. In the middle of S. chuatsi CL region, a microsatellite sequence (AGC)(6-8) was found, which is also present in another perciform species, the spotted wolffish (Anarhichas minor). The comparison of amino acid sequence of the mandarin fish CL domain with those of other vertebrates showed the highest degree of similarity of 94.5% to the spotted wolffish, while the similarity with rainbow trout (Oncorhynchus mykiss) Ig L1 (62.7%) and channel catfish (Ictalurus punctatus) Ig LG (55.9%) isotypes is also higher. However, there is only 50% identity in the VL regions between the mandarin fish and the wolffish. The sequence similarity of the mandarin fish CL domain with those of higher vertebrate did not readily allow it to be classified as kappa or lambda isotype. The phylogenetic analyses also demonstrated that the CL genes of the mandarin fish and most other teleost fish cluster as a separate branch out of the mammal kappa and lambda branches. (C) 2003 Elsevier B.V. All rights reserved.

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The unicellular cyanobacterium Synechocystis sp. PCC6803 can grow heterotrophically in complete darkness, given that a brief period of illumination is supplemented every day (light-activated heterotrophic growth, LAHG), or under very weak ( < 0.5 mumol m(-2) s(-1)) but continuous light. By random insertion of the genome with an antibiotic resistance cassette, mutants defective in LAHG were generated. In two identical mutants, sll0886, a tetratricopeptide repeat (TPR)-family membrane protein gene, was disrupted. Targeted insertion of sll0886 and three downstream genes showed that the phenotype was not due to a polar effect. The sll0886 mutant shows normal photoheterotrophic growth when the light intensity is at 2.5 mumol m(-2) s(-1) or above, but no growth at 0.5 mumol m(-2) s(-1). Homologs to sll0886 are also present in cyanobacteria that are not known of LAHG. sll0886 and homologs may be involved in controlling different physiological processes that respond to light of low fluence. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

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This paper reports the mechanical properties and fracture behavior of silicon carbide (3C-SiC) thin films grown on silicon substrates. Using bulge testing combined with a refined load-deflection model of long rectangular membranes, which takes into account the bending stiffness and prestress of the membrane material, the Young's modulus, prestress, and fracture strength for the 3C-SiC thin films with thicknesses of 0.40 and 1.42 mu m were extracted. The stress distribution in the membranes under a load was calculated analytically. The prestresses for the two films were 322 +/- 47 and 201 +/- 34 MPa, respectively. The thinner 3C-SiC film with a strong (111) orientation has a plane-gstrain moduli of 415 +/- 61 GPa, whereas the thicker film with a mixture of both (111) and (110) orientations exhibited a plane-strain moduli of 329 +/- 49 GPa. The corresponding fracture strengths for the two kinds of SiC films were 6.49 +/- 0.88 and 3.16 +/- 0.38 GPa, respectively. The reference stresses were computed by integrating the local stress of the membrane at the fracture over edge, surface, and volume of the specimens and were fitted with Weibull distribution function. For the 0.40-mu m-thick membranes, the surface integration has a better agreement between the data and the model, implying that the surface flaws are the dominant fracture origin. For the 1.42-mu m-thick membranes, the surface integration presented only a slightly better fitting quality than the other two, and therefore, it is difficult to rule out unambiguously the effects of the volume and edge flaws.

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A fiber Bragg grating (FBG) pressure sensor packaged by using a hard core in the membrane is presented. By utilizing the unique membrane-based FBG packagine method, its pressure sensitivity has been effectively enhanced. The pressure sensitivity of the FBG reaches 5.75 X 10(-3)/MPa within the pressure range of 0.0.16 Mpa. (C) 2009 Wiley Periodicals, Inc. Microwave Opt Technol Lett 51 1279-1281, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mop.24335

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A Si resonant-cavity-enhanced (RCE) photodiode was fabricated on a silicon membrane. The Si membrane was formed by etching from the back side of the silicon-on-insulator substrate with the buried SiO2 layer as etch-stop layer. A gold layer was deposited serving as an electrode layer and bottom mirror of the RCE photodiode. The photodiode had an external quantum efficiency of 33.8% at the resonant wavelength of 848 nm and a full width at half maximum (FWHM) of 17 nm. The responsivity was 4.6 times that of a conventional Si p-i-n photodiode with the same absorption layer thickness. (c) 2005 American Institute of Physics.

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Si-based membrane RCE photodetectors were introduced. The RCE photodiodes were fabricated on silicon membranes formed from SOI substrate. Compared with the conventional p-i-n photodiode, the responsivity has a threefold enhancement.

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BRUNOL/CELF家族RNA结合蛋白在转录后调控(post-transcriptional regulation)中起着至关重要的作用,参与多种组织的发育过程。本研究中,我们描述了非洲爪蟾5个Brunol 基因的克隆与表达。其中只有Brunol2是母源性以及合子表达的,其它的4个Brunol基因都是合子表达的,但起始表达的发育时期有所不同。爪蟾发育过程中,Brunol1、4和5基因特异性地在神经系统中表达,包括脑、脊髓、眼泡和耳泡。Brunol2和3基因在体节中胚层与神经系统表达。Brunol2也在晶状体中有非常高的表达。在转染的Hela细胞中,BRUNOL1、2和3蛋白定位于细胞质和细胞核中,BRUNOL4和5只是定位于细胞质中,显示它们具有不同的功能。 人的microcephalin1(MCPH1)基因的遗传突变产生原发性小头症,而在人类的进化过程中,这个基因的变化可能对人脑体积的增加和认知能力的增强也起到重要的作用。但是对于MCPH基因在其它物种中功能的研究才刚刚开始。我们克隆了非洲爪蟾MCPH基因,发现非洲爪蟾具有A,B两个同源基因,其功能域的保守性较高,暗示非洲爪蟾MCPH基因仍然执行一些保守的功能,但MCPHB由于突变只编码一种截短的蛋白,目前尚不清楚它是否是有功能的。胚胎原位杂交的结果显示MCPHA,B基因在胚胎发育中的表达图式相似,但MCPHB的表达水平较低。在神经胚期,二者均表达于头部基板区,在尾芽期主要表达于咽鳃区,而在脑区的表达并不显著,与小鼠中的表达模式不同,提示在爪蟾中MCPH基因可能主要参与咽鳃区而不是脑的发育。 为了进一步筛选这些蛋白可能的结合因子,我们构建了非洲爪蟾双杂交cDNA文库。其中利用修饰的随机引物和特别设计的连接头在合成双链cDNA时,在下游合成一个SalI限制性酶切位点,可以将cDNA定向插入载体。通过对于空克隆率和插入片段长度等一系列参数的分析,表明这个定向cDNA文库的构建是成功的。

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In this paper, an interdigital electrode lipid film odour sensor (ILOS) is designed, fabricated and tested. It is made from a microfabricated chemiresistor coated with a synthetic multibilayer film. Nine odorants in gas phase at room temperature have been detected using the odour sensor. For most of the odorants, the relation between the response of the ILOS and odorant concentration obeys Stevens' power law, and there is a good correlation between the minimum odorant concentrations that give rise to a change of the sensor's conductance and human olfactory thresholds.