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根据已发表的序列,分别在鸡贫血病毒(CAV)环形基因组DNA(全长2.3kb)的EcoRI位点和BamHI位点的两侧选择适当序列合成两对引物,用PCR技术,从斑点杂交检测到病毒核酸的CAV感染的MDCCRP1细胞基因组DNA中,分别扩增出包含EcoRI和BamHI分割开的病毒基因组两部分(1.5kb和0.8kb)约1.5kb和约1.25kb的两个片段。再将其中相应序列拼接克隆进pUC18载体,获得包含CAV全基因组序列DNA片段的克隆质粒pCAV2.4。酶切分析表明,该质粒具有预期的BamHI位点、PstI位点、HindⅢ位点,而预期的EcoRI位点消失。重组质粒插入DNA片段的两端序列分析表明,质粒pCAV2.4是包含CAV全基因组序列的重组质粒,插入DNA片段序列中的EcoRI位点序列发生了一个碱基突变。 相似文献
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限制性内切酶BamHI水解中国野生葡萄葛lei的叶绿体DNA(cpDNA),电泳分离为34条带,最大为11.7kb,最小为0.23kb,分别为pUC和pBR322质粒克隆所得片段,转化大肠杆菌,并从转化菌中提取质粒DNA,经酶切电泳分析证明含有葛lei cpDNA经BamHI水解的不同片段,从而构建了葛lei cpDNA的BamHI质粒文库。 相似文献
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环状芽孢杆菌C-2几丁酶基因的克隆 总被引:4,自引:0,他引:4
环状芽孢杆菌(Baciluscirculans)C2总DNA经PstI部分酶切后分离2~10kb的片段,插入质粒pUC19的PstI位点,转化大肠杆菌(Escherichiacoli),利用几丁质平板从约8000个重组子中筛选到一个几丁酶基因阳性克隆(命名为pCHT1)。用12种限制酶对重组质粒进行的酶切分析表明,重组质粒中的插入片段长30kb,其中各有一个KpnI,SacI和SspI位点。把该克隆片段反向插入pUC19的PstI位点所得到的重组子同样具有几丁酶基因表达活性,说明此片段含有一个完整的几丁酶基因,其自身的启动子能被大肠杆菌转录系统所识别。Southern杂交证实了该片段来自于B.circulansC2基因组,且以单拷贝形式存在,它不能与来自于其它7株几丁酶产生菌的总DNA杂交。 相似文献
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丙肝病毒融合抗原基因NS3-C定点整合入衣藻叶绿体基因组的研究 总被引:21,自引:1,他引:21
用PCR 方法从丙型肝炎病毒(HCV) cDNA 文库中克隆了两段DNA 片段,即HCV 基因组非结构NS3区抗原基因(约0.7 kb)和核心抗原C区抗原基因(约0.6 kb)的cDNA 片段。在两段cDNA 间加入连接肽Ser- Pro- Gly- Ser 的密码子序列,构建成融合抗原基因NS3- C。将该融合基因与衣藻叶绿体基因atpA 的启动子和rbcL 基因的3′末端连接,得到丙肝病毒融合抗原基因NS3- C表达盒,再将该表达盒与选择标记基因aadA 表达盒和衣藻叶绿体基因组同源片段连接,构建成衣藻叶绿体转化载体pSS6。基因枪法转化衣藻叶绿体,经壮观霉素筛选获得转化再生的单藻落,对转基因衣藻的PCR 和Southern 杂交分析表明,融合抗原基因NS3- C已整合到衣藻叶绿体基因组中。 相似文献
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对含有麦迪霉素4″-O-丙酰基转移酶(mpt)基因的BamHI-BamHI8.0kb的DNA片段进行限制性酶切分析,绘制出了含有21个酶切位点的限制性酶切图谱。以含有碳霉素异戊酰基转移酶基因(CarE)的2.4kb DNA片段为探针,经Southern blot分子杂交,将mpt定位于一个EcoRI-EcoRI-PstI3.0kb的DNA片段上,将该片段克隆至大肠杆菌/链霉菌穿梭质粒载体pWHM3 相似文献
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杂合质粒pIJ8310是链霉菌低拷贝质粒pIJ9221携带了10.8kb来自变铅青链霉菌JT46菌株,编码对噬菌体ΦHAU3抗性的DNA片段。已知Tn4560插到该克隆中3kb的EcoRI或3.3kb的BamHI片段(这两个片段之间有2.5kb的重叠区)上,中断了ΦHAU3抗性基因的表达。已将3.0kb的BcoAI片段分别以两种不同的取向克隆到pBluescriptSK(+)上。核苷酸序列分析揭示出一个1.3kb的ORF的存在,其所编码的478个氨基酸组成的蛋白与λ噬菌体的ea59基因所编码的氨基酸序列显示了高度的同源性,其氨基酸序列的同一性高达37%,相似性达58%,而λ噬菌体的ea59基因是编码赖ATP和单键DNA的核酸内切酶I的,该区域与λ的复制无关。此外,这段具有高度同源性的DNA区域的G+C%只有60%,低于变铅青霉菌DNAG+C%的平均值(74%)。 相似文献
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点状产气单胞菌脯氨酰内肽酶基因克隆与序列测定 总被引:2,自引:0,他引:2
点状产气单胞菌点状亚种(Aeromonas puncata subsp.punctata)具有脯氨酰内肽酶(prolyl endopeptidase PEP)活性。将其染色体DNA有Eco RⅠ部分酶切后回收8-16kb的DNA片段,与EcoRⅠ消化载体pUC18连接后转化E.coil DH5α,用该酶的专一性底物Benzyloxycarbonyl-Gly-β-naphthylamide从质粒库中 相似文献
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用AcNPVp74基因3’端的1.4kb片段,通过Southernblotting将SpltNPV的p74基因定位于XhoI(4.9kb)、EcoRI(4.4kb)、BamHI(3.0kb)片段上。进一步用ExoⅢ和构建亚克隆测序法,对长2545bp的p74基因进行,其中1974bp的编码编码658个氨基酸,蛋白分子量约75.87kD,其编码蛋白与AcMNPV和CfMNPVp74蛋白的氨基酸序列同 相似文献
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人巨细胞病毒的分子克隆及其特异性DNA探针的制备 总被引:6,自引:0,他引:6
从人巨细胞病毒(HCMV)培养物中提取HCMV并抽提其DNA,经限制性内切酶BamHI完全消化后,与质粒pBluescript-SK重组建立了HCMV的DNA文库,从此文库。中随机筛选出两个重组质粒(pCMV-1和pCMV-2),用BamHI分析证明其中所含的病毒DNA片段的大小分别为1.0kb和7.5kb,将这两种质粒大量扩增纯化后,用光生物素进行标记作为探针,证明其只与HCMV反应,与正常人细胞DNA及Ⅰ型和Ⅱ型单纯疤疹病毒DNA无交叉反应。 相似文献
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In experiments on Black Sea skates (Raja clavata), the potential of the receptor epithelium of the ampullae of Lorenzini and spike activity of single nerve fibers connected to them were investigated during electrical and temperature stimulation. Usually the potential within the canal was between 0 and –2 mV, and the input resistance of the ampulla 250–400 k. Heating of the region of the receptor epithelium was accompanied by a negative wave of potential, an increase in input resistance, and inhibition of spike activity. With worsening of the animal's condition the transepithelial potential became positive (up to +10 mV) but the input resistance of the ampulla during stimulation with a positive current was nonlinear in some cases: a regenerative spike of positive polarity appeared in the channel. During heating, the spike response was sometimes reversed in sign. It is suggested that fluctuations of the transepithelial potential and spike responses to temperature stimulation reflect changes in the potential difference on the basal membrane of the receptor cells, which is described by a relationship of the Nernst's or Goldman's equation type.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. I. M. Sechenov, Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Pacific Institute of Oceanology, Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Neirofiziologiya, Vol. 12, No. 1, pp. 67–74, January–February, 1980. 相似文献
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N. P. Vesselkin Yu. V. Natochin 《Journal of Evolutionary Biochemistry and Physiology》2010,46(6):592-603
Evolution of living organisms is closely connected with evolution of structure of the system of regulations and its mechanisms.
The functional ground of regulations is chemical signalization. As early as in unicellular organisms there is a set of signal
mechanisms providing their life activity and orientation in space and time. Subsequent evolution of ways of chemical signalization
followed the way of development of delivery pathways of chemical signal and development of mechanisms of its regulation. The
mechanism of chemical regulation of the signal interaction is discussed by the example of the specialized system of transduction
of signal from neuron to neuron, of effect of hormone on the epithelial cell and modulation of this effect. These mechanisms
are considered as the most important ways of the fine and precise adaptation of chemical signalization underlying functioning
of physiological systems and organs of the living organism 相似文献
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