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以我国栗疫菌[Cryphonectria(=Endothia)parasitica]低毒力菌株EpC140(广西)的[γ─ ̄32P]ATP末端标记dsRNA作探针,与5种真菌病毒dsRNA进行分子杂交。探针可与紫孢侧耳病毒(Pleurotussepidusvius)和小麦全蚀菌病毒(Gaeuman─nomycesgraminis·virus)的dsRNA杂交,但不能与黑曲霉病毒(Aspergillusnigervirus)、产黄青霉病毒(Penicilliumchrysogenumvirus)和稻瘟菌病毒(Pyriculariaoryzaevirus)的dsRNA杂交。当以低毒力菌株EpC32(云南)作探针时,结果相同。 相似文献
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dsRNA介导的RNA干扰 总被引:5,自引:0,他引:5
在多种生物中 ,外源或内源性的双链RNA(double strandedRNA ,dsRNA)导入细胞中 ,与dsRNA同源的mRNA则受到降解 ,因而其相应的基因受到抑制。这种转录后基因沉默 (post transcriptionalgenesilencing ,PTGS)机制首先在线虫 (C .elegans)中得以证实。由于这是一种在RNA水平的基因表达抑制 ,故也称为RNA干扰 (RNAinterfer ence) ,简称RNAi[1] 。随后发现 ,在各种生物 ,如果蝇[2 ] 、拟南芥菜[3 ] 、及小鼠[4] 等均存在dsRNA介导… 相似文献
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以我国栗疫菌(Cryphonectria(=Endothia)parasitica)低毒力菌株EpC140(广西)的(γ-^32P)ATP末端标记dsRNA作探针,与5种真菌病毒dsRNA进行分子杂交。探针可与紫孢侧耳病毒(Pleurotus sapidus virus)和小麦全蚀菌病毒(Gaeuman-nomyces graminis.virus)的dsRNA杂交,但不能与黑曲霉病毒(Pyric 相似文献
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香菇病毒HKB(Lentinula edodes mycovirus HKB,LeV)是一种具有潜隐性特点的真菌病毒,广泛存在于香菇菌株中。为了快速、准确地检测出LeV,根据LeV病毒基因组序列(AB.429556.2)的信息,设计合成一对引物,对25个香菇菌株进行RTPCR检测,在20个香菇菌株中分别扩增出LeV病毒基因组特有的条带,在5个香菇菌株中未能扩增出条带。通过对25个香菇菌株进行dsRNA提取检测,结果也表明不存在扩增片段的菌株提取不到dsRNA,存在扩增片段的菌株能够提取得到dsRNA。因此建立的RT-PCR方法可以快速检测到香菇dsRNA病毒LeV,能够对香菇的质量检测提供技术支持。 相似文献
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采用生物学测定和凝胶电泳的方法,对中国东部栗疫病菌(Cryphonectria parasitica)的dsRNA病毒进行了研究。根据电泳图谱,中国东部栗疫病菌的dsRNA病毒可以分为5种类型,其中类型1只具有一个12.7kh的条带,占大多数;类型Ⅱ有两条带,一条12.7kh,另一条5.2kb左右,只有一个菌株354;类型Ⅲ则除了12.7kb的条带外,还有3条小于2kb的小片段,有238和250两个菌株;dsRNA属于类型Ⅳ的为269和344两个菌株,它只含有两个1.8—3.1kb的小片段;dsRNA类型Ⅴ只有一个菌株280,除了一条12.7kb分子外,还有4条2.6—3.3kb的小片段。含有dsRNA的菌株培养性状多样,根据菌落特征.栗疫病菌可以划分为5种培养类型,但菌株的培养类型与dsRNA的类型间没有对应的关系。毒力测定表明含有dsRNA病毒的供试菌株大多属于低毒力类型; 相似文献
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Sequences hybridizing to mRNA, oligo(dT) and dsRNA from pre-mRNA are contiguous in the cloned mouse DNA fragments. 总被引:1,自引:0,他引:1 下载免费PDF全文
O N Tokarskaya N A Tchurikov P L Ivanov D A Kramerov A P Ryskov 《Nucleic acids research》1980,8(3):425-440
Fragments from the DNA of mouse embryos produced by restriction endonucleases HindIII were cloned in pBR322 plasmid and examined for the ability to hybridize in situ with [32P] labeled cDNA synthesized from the polysomal poly(A)+mRNA template. Several of the selected clones were examined for the presence of specific sequences inside the cloned mouse DNA fragments by the blotting procedure of southern [1]. The data obtained indicate that the majority of the cloned mouse DNA fragments contained sequences hybridizing with cDNA, oligo(dT) and double-stranded regions from pre-mRNA. The results of hybridization experiments and double digestion with HindIII+HaeIII endonucleases provide evidence that these sequences could be contiguous in the given restriction DNA fragments. 相似文献
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A fragment of the α-fetoprotein (AFP) structural gene was purified and amplified by bacterial cloning techniques. Double-stranded DNAAFP was constructed from a cDNA copy of greater than 95% pure mRNAAFP and inserted into E. coli plasmid pBR322 by poly(dA-dT)-linkers. Chimeric plasmid DNA isolated from transformants of E. coli strain χ1776 have been shown to contain α-fetoprotein sequences by hybridization to labeled mRNAAFP. One clone, designated pA5 (chimeric plasmid pBR322 containing a cDNAAFP sequence isolated from clone 5), has been studied in more detail. The inserted sequence of approximately 950 nucleotide pairs was positively identified by a hybridization-translation procedure. Hybridization of [3H]uridine-labeled poly(A)-containing RNA from an AFP-secreting cell line to excess pA5 DNA immobilized on nitrocellulose filters was used to show the selectivity of this probe for detecting expression of the AFP gene. 相似文献
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Polyadenylated RNA was isolated from acute-phase liver and transcribed into cDNA. After homopolymer tailing this was cloned into the PstI site of pBR322 which was used to transform E. coli RR1 cells. Clones containing cDNA corresponding to acute-phase proteins were identified by differential hybridization with 32P-labelled normal and acute-phase cDNA. A clone synthesizing about 10 ng of alpha 1-acid glycoprotein per E. coli colony was detected using a solid-phase based immunochemical procedure. The identification of the clone was verified by competition assay with authentic alpha 1-acid glycoprotein isolated from serum and by restriction analysis of the cDNA inserted into pBR322. 相似文献
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A recombinant plasmid (C357; 3.5 Mdal) containing heterologous DNA (pBR322 [2.6 Mdal] with cDNA for an egg yolk protein fromDrosophila grimshawi) inEscherichia coli strain HB101 survived in and was recovered on selective media from sterile and nonsterile soil during 27 days at frequencies similar to those of theE. coli(pBR322) system. In sterile saline, the numbers of all cells decreased during 34 days, but the numbers of the plasmidless host declined less. There was no selective loss of the heterologous DNA in either soil or saline, as determined by colony hybridization with a32P-labeled DNA probe for the cDNA, but the HB101(C357) appeared to be less able than HB101(pBR322) to cope with conditions of starvation. These results suggested that nonessential eucaryotic DNA inserted into plasmid DNA has little effect on the survival in soil or saline of the bacterial host and the maintenance of the vector. 相似文献
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Bacteriophage lambda gt11 has been used quite extensively for producing cDNA libraries. The cDNA inserts are usually subcloned into a plasmid vector for large scale production and analysis. However, isolating the recombinant DNA of interest from the phage clones can be a tedious task. Since the E. coli strain Y1088 used for lambda gt11 phage infection carries a pBR322-derived plasmid endogenously, we reasoned that this endogenous plasmid could be used directly for cloning the cDNA phage insert. In this report, we describe a method in which cDNA inserts from lambda gt11 phage were cloned directly into the pBR322 plasmid vector, bypassing the time-consuming procedures of preparing plasmid DNA as a subcloning vector. This method is likely to be extended to the cloning of DNA inserts derived from other phage lambda vectors when bacteria containing endogenous pBR322 are used as host cells. 相似文献
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Cloning and expression of the cDNA coding for aequorin, a bioluminescent calcium-binding protein 总被引:8,自引:0,他引:8
D Prasher R O McCann M J Cormier 《Biochemical and biophysical research communications》1985,126(3):1259-1268
Aequorin is a bioluminescent protein which consists of a polypeptide chain (apoaequorin), coelenterate luciferin, and bound oxygen. Aequorin produces blue light upon binding Ca2+. We have isolated six recombinant pBR322 plasmids which contain apoaequorin cDNA sequences. A mixed synthetic pBR322 plasmids which contain apoaequorin cDNA sequences. A mixed synthetic oligonucleotide probe was used to identify these cDNAs. An extract of an E. coli strain possessing the largest cDNA contained apoaequorin. This apoaequorin can be converted to aequorin in the presence of coelenterate luciferin, 2-mercaptoethanol, and O2. This cDNA is therefore apparently full-length. 相似文献
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Cloning of an almost full-length chicken conalbumin double-stranded cDNA. 总被引:17,自引:6,他引:11 下载免费PDF全文
M Cochet F Perrin F Gannon A Krust P Chambon G S McKnight D C Lee K E Mayo R Palmiter 《Nucleic acids research》1979,6(7):2435-2452
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Auday Maki Salwa Atwan Janan Al-Kaledar Andrew Beaman Robert Skoff 《Biotechnic & histochemistry》1997,72(1):38-44
Technical limitations are associated with conducting successful in situ hybridization. In this study, three cell types including a tumor neuroblastoma cell line (Neuro-2a), an oligodendrocyte primary culture, and a nonneuronal acute lymphoblastic leukemia cell line (Reh) were used to conduct successful nonradioactive in situ hybridization. Two cDNA probes were used. A 1 kb probe was used to identify the expression of proteolipid protein (PLP) mRNA in a primary culture of oligodendrocytes. A 760 bp cDNA was used to identify the expression of ubiquitin C-terminal hydrolase (UCH-L1) mRNA in Neuro-2a and Reh cells. The probes were labeled with digoxigenin-11-dUTP, denatured, and hybridized with cells fixed on coverslips. The efficiency of the labeling was tested using dot blot analysis by comparing the intensity of our labeled probes with known concentration of the probe labeled by the provider. The nonspecific signals were washed off, followed by detection of a signal specific to the gene. The specificity of the probes was determined by treating the cells with RNase A, hybridizing with bacterial Dig-labeled cDNA (pBR322) and hybridizing the tissues in the absence of labeled probe. During the labeling step, we found that addition of co-precipitants, such as tRNA or glycogen, during precipitation of the labeled probe followed by overnight incubation at -20 C is essential for good recovery of labeled cDNA. Dissolving the labeled probe in a buffer solution containing sodium dodecyl sulfate improves the quantity of the labeling. At the cellular level, prehybridization treatments optimize the permeability of the cell and allow efficient penetration of the labeled probe. Fixing with paraformaldehyde or an ethanol-acetic acid mixture can preserve the structure of cultured cells. To increase the signal to noise ratio, cells were treated with 0.2 N HC1 followed by extensive washes using a solution with a high salt concentration and containing dextran sulfate. This treatment significantly improves the signal and reduces the background in cell cultures, but not in tissue sections. The ability to reuse the labeled probe-hybridization mixture is another advantage for using nonradioactive in situ hybridization. 相似文献