Ingested foreign (phage M13) DNA survives transiently in the gastrointestinal tract and enters the bloodstream of mice

Is the epithelial lining of the mammalian gastrointestinal (GI) tract a tight barrier against the uptake of ingested foreign DNA or can such foreign DNA penetrate into the organism? We approached this question by pipette-feeding circular or linearized double-stranded phage M13 DNA to mice or by adding M13 DNA to the food of mice whose fecal excretions had previously been shown to be devoid of this DNA. At various post-prandial times, the feces of the animals was tested for M 13 DNA sequences by Southern or dot blot hybridization or by the polymerase chain reaction (PCR). On Southern blot hybridization, the majority of M13 DNA fragments were found in the size range between < 200 and 400 by (base pairs). For the PCR analysis, synthetic oligodeoxyribonucleotide primers were spaced on the M13 DNA molecule such that the sizes of the persisting M13 DNA fragments could be determined. We also extracted DNA from whole blood or from sedimented blood cells of the animals at different times after feeding M t3 DNA and examined these DNA preparations for the presence of M13 DNA by dot blot hybridization or by PCR. M13 DNA fragments were found between 1 and 7 h postprandially in the feces of mice. By PCR analysis, fragments of 712, 976, and 1692 by in length were detected. In DNA from blood, M13 DNA fragments of up to 472 by were found by PCR between 2 and 6 h after feeding. Dot blot or Southern blot hybridization revealed M13 DNA at 2 and 4 h, but not at 1, 8 or 24 h after feeding. This DNA was shown to be DNase sensitive. M13 DNA was found both in blood cells and in the serum. A segment of about 400 by of the DNA amplified by PCR from feces or blood was analyzed for its nucleotide sequence which was found to be identical to that of authentic M13 DNA, except for a few deviations. M13 DNA could not be detected in the feces or in the blood of the animals prior to feeding or prior to 1 h and later than 7 h after feeding. These controls attest to the validity of the results and also argue against the possibility that the murine GI tract had been colonized by phage M13. Moreover, M13 DNA-positive bacterial colonies were never isolated from the feces of animals that had ingested M13 DNA. The results of reconstitution experiments suggested that 2 to 4% of the orally administered M13 DNA could be detected in the GI tract of mice. A proportion of about 0.01% to 0.1% of the M13 DNA fed could be retrieved from the blood.     

用地高辛标记探针检测基因工程人β干扰素制品中的外源DNA

基因工程人β干扰素工程菌经发酵培养、纯化后获得纯的制品,采用斑点杂交技术,用非放射性地高辛标记超声裂解的全工程菌ＤＮＡ作为探针,检测基因工程人β干扰素纯品,结果显示：基因工程人β干扰素纯品中的外源ＤＮＡ含量小于１００ｐｇ／剂。     

秀丽隐杆线虫经福尔马林处理后上调表达基因的分析

秀丽隐杆线虫(Caenorhabditis elegans)是一种重要的模式生物,目前已经被广泛应用到生物对各种驱虫剂抗性机制的研究中。福尔马林被普遍用于鱼类寄生虫病的防治中,但是由于长期的应用,许多寄生虫对它产生了一定的抗药性。研究以秀丽隐杆线虫为研究对象,分析了它在福尔马林刺激下基因表达的改变情况。结果显示,经福尔马林处理后差异表达的有676个克隆,通过斑点杂交技术进一步筛选,对其中差异显著的161个克隆进行了测序分析。测序结果经BLAST分析发现:(1)细胞凋亡相关基因的表达发生上调,这些基因编码包括线粒体呼吸链相关蛋白、含TPR序列的蛋白SGT-1、热休克蛋白、氧化应激相关蛋白、胞吞过程相关蛋白、DNA复制和修复相关蛋白以及其他一些重要的凋亡相关蛋白;(2)编码重要的转录调节因子和信号转导相关蛋白如转录激活因子FosB/c-Fos、G蛋白、细胞周期蛋白B、钙结合蛋白、核小体装配蛋白NAP-1等的基因的表达发生上调;(3)能量代谢和蛋白质、脂肪、氨基酸代谢途径中的有些基因的表达也发生上调;(4)除了以上已知功能的基因外,还有一些未命名蛋白质基因。这说明福尔马林对秀丽隐杆线虫的影响除了诱导细胞凋亡之外,还影响其他细胞代谢活动的改变。实验为进一步研究生物体对福尔马林的抗性机制奠定了一定的基础。       

实验性石棉肺病变中Ⅰ型及Ⅲ型胶原基因表达的研究

以斑点杂交法测定染溫石棉尘后30天与60天大鼠肺组织中前胶原mRNA的水平,即proα_1(Ⅰ)、proα_2(Ⅰ)、proα_1(Ⅲ)mRNA的水平,并与正常大鼠肺组织对比,结果显示,染石棉尘的肺组织中这三种mRNA显著地高于正常对照组。在染尘后60天时Ⅰ型胶原的两种mRNA仍呈上升趋势,而Ⅲ型胶原的mRNA则呈稳定状态。体外实验的结果表明溫石棉及青石棉纤维都可以刺激2BS细胞中Ⅰ、Ⅲ型胶原基因的表达。矽肺组织来源的致纤维化因子与石棉纤维都具有促进胶原基因表达的作用,其共同作用效果更強。证明这两种因素都参与调节石棉肺的纤维化作用。     

Characterization of isolates and clones of leishmania by analysis of kinetoplast DNA

The genetic characterization of pathogenic isolates of Leishmania was attempted by analysis of the molecular properties of kinetoplast DNA (kDNA) minicircles. Unit minicircle size is not conserved during speciation of Leishmania since the minicircles of strains and clones of L t major are smaller (700 bp) than those found in certain strains of L mexicana ssp (820 bp), L donovani (850 bp) or L t tropica (900 bp). Schizodeme analysis of minicircles reveals a high degree of sequence divergence in kDNA of Leishmania with the degree of microheterogeneity varying between species. This sequence divergence allows the discrimination of species, strains, and clones of Leishmania into schizodemes. Southern blot hybridization experiments reveal that at high stringency overall minicircle sequence homology is conserved among clones and strains of one species (L t major) but not between different species. This property of minicircle DNA permits the use of kDNA probes as a species-specific diagnostic test for the identification of unknown Leishmania isolates. The properties of kDNA from an L t tropica strain LRC-L32 (a “recidiva” organism) are so diverged from those of L t major strains as to support the classification [22,23] of L t tropica and L t major as separate species of Leishmania rather than subspecies of L tropica.     

PCR和生物素探针对HFRSV的检测和分型的探讨

分析比较肾综合征出血热病毒（ＨＦＲＳＶ）７６／１１８株和Ｒ＿（２２）株的核苷酸序列,根据引物设计原则及检测分型的目的,设计并合成了３对引物。１对引物取于两毒株间的高同源区段,作为共同引物和外引物;另两对引物取于两毒株间的低同源区段,分别作为野鼠型引物、家鼠型引物和内引物。建立了ＤＮＡ聚合酶链反应（ＰＣＲ）和ＮｅｓｔＰＣＲ方法,并用ＮｃｓｔＰＣＲ合成了两种型特异的生物素探针。ＰＣＲ检测７６／１１８、Ａ＿９、陈、Ｒ＿２、Ｒ＿２２５个毒株,用外引物时均扩增出１条约３００ｂｐ的条带;用内引物的野鼠型引物时,除Ｒ＿（２２）株之外,其余４株均扩增出１条约７０ｂｐ的条带。斑点杂交试验证实了ＰＣＲ检测分型的准确性。ＮｅｓｔＰＣＲ和生物素探针斑点杂交试验可以测出１－１０ｂｇ的目的ｃＤＮＡ。     

Expression and cellular localization of the mRNA for the 25-kDa heat-shock protein in the mouse

The 25-kDa heat-shock protein (Hsp25) is a member of the small heat-shock protein family but its function remains largely unknown. In the present study we examined the expression and cellular localization of Hsp25 mRNA in mice under physiological, unstressed conditions using Northern blot and in situ hybridization analyses with specific oligonucleotide probes. At the organ level, high amounts of Hsp25 mRNA were detected in the oesophagus, skin,eye, stomach, lung and urinary bladder, with moderate amounts in the heart, skeletal muscle, aorta, adrenal gland, ovary, testis, uterus, large intestine, and thymus. At the cellular level, intense to moderate signals for Hsp25 mRNA were localized in the muscle cells of smooth, heart and skeletal types, in the epithelial cells of stratified squamous and transitional types and of the oviduct, in the steroid endocrine cells of the adrenal cortex and corpus luteum, as well as in the spermatocytes of the testis. In contrast, the signal was scarcely detectable in the nervous tissues, lymphatic tissues, the columnar epithelial cells of the digestive tract, or the parenchymal cells of the liver, pancreas and kidney. These results suggest some significant role for Hsp25 in distinct populations of mouse cells under physiological conditions.     

基于DIG -化学发光法的Southern blot方法优化

目前,基于DIG-化学发光法的Southern blot技术已在生物实验室广泛应用,但在试验过程中经常会遇到背景黑、目的信号弱等问题,以致不能获得理想结果.在棉花线拉体基因组RFLP分析过程中结合前人经验对基于DIG -化学发光法的Southern blot技术在探针标记、探针浓度、探针剥高等方面进行了优化.优化后的方...     

A new method for rapid screening of bacterial species- or subspecies-specific DNA probes

A simple assay for the rapid screening of bacterial species- or subspecies-specific DNA probes for the random cloning method is presented, involving the use of genomic DNAs as probes and recombinant plasmid DNAs containing genomic DNA digested with HindIII as targets. The optimal amount of target DNAs and the concentration of digoxigenin-labeled genomic DNA probes were 20 ng and 100 ng ml(-1) (or 10 ng and 200 ng ml(-1)), respectively. The method was applied to the development of Fusobacterium nucleatum subspecies-specific probes. Our results showed that four out of 96 probes were F. nucleatum subspecies-specific, which was confirmed by Southern blot analysis. Our results indicate that the new method can be used for the rapid screening of species- or subspecies-specific probes.     

异羟基洋地黄毒甙元(Digoxigenin)标记的RNA探针在丁型肝炎病毒核酸杂交中的应用

应用异羟基洋地黄毒甙元(Digoxigenin)标记的RNA探针,检测了人和黑猩猩血清及肝脏中丁型肝炎病毒核酸,并与~(32)P标记同一探针做了比较。结果表明,异羟基洋地黄毒甙元标记的RNA探针的杂交效果与同位索探针一致(同源cDNA0.2pg),可用于人和动物血清及肝脏标本内HDV核酸的检测。     

DNA analysis of intracellular campylobacter-like organisms associated with the porcine proliferative enteropathies: novel organism proposed

Intracellular Campylobacter-like organisms are a consistent feature of the porcine proliferative enteropathies. The relationship between these organisms and known Campylobacter sp. previously associated with the disease was studied using restriction enzyme analysis and DNA-DNA blot hybridization techniques. BglII restriction enzyme fragment patterns of DNA of the Campylobacter-like organisms were fundamentally different from those of C. mucosalis, C. hyointestnalis, C. jejuni, and C. coli. Crude DNA preparations from Campylobacter-like organisms hybridized strongly with homologous preparations, weakly with porcine DNA and not at all with DNA from Campylobacter sp. Fragment specific DNA probes prepared from Campylobacter-like organisms only hybridized with homologous preparations. This work suggests that the intracellular Campylobacter-like organisms are not one of the known Campylobacter sp. It is possible that they are a novel, uncultured organism worthy of a new name, such as HC. intracellulare'.     

Rapid RAPD screening of plant DNA using dot blot hybridization

Scoring of the results of RAPD analysis using gel electrophoresis imposes a constraint on throughput. To circumvent this barrier, dot-blot hybridization was substituted for electrophoresis. Arbitrarily amplified fragments from barley and wheat genomic DNA were labelled and used as probes for the identification of identical fragments in subsequent amplification reactions. None of the twelve fragments used as probes exhibited significant levels of croos-hybridization to other fragments amplified by the same arbitrary primer. The strength of the hybridization signal facilitates more accurate and more sensitive detection of diagnostic fragments than gel electrophoresis. In addition, the defined spatial orientation (microtitre dish format) of the ± results provide an excellent format for automated data collection. The use of dot blot hybridization to analyse PCR products well decrease the cost and time requirements of marker-assisted selection. This technique will also facilitate the rapid application of PCR-based maps.     

诺如病毒RT-PCR-反向斑点杂交检测方法的建立

旨在建立诺如病毒RT-PCR-反向斑点杂交检测方法。选取诺如病毒较为保守的RdRp基因作为扩增对象,经RT-PCR扩增后将目的片段克隆到pGEM-T载体中。以重组质粒为模版,选择合成寡核苷酸探针及生物素标记引物。生物素标记引物的扩增产物经热变性后与固定在硝酸纤维素膜上的探针进行杂交反应,经显色后判定结果。出现明显的蓝紫色斑点为诺如病毒阳性,如无斑点则为阴性。对5份临床样品进行检测,并以RT-PCR对比验证。结果显示,利用反向斑点杂交法对重组质粒的检测限为100拷贝/μL,在5例实际样品检测中有1例为阳性,与RT-PCR判定结果一致。建立了诺如病毒的RT-PCR-反向斑点杂交检测方法,该方法特异性好,灵敏度高,操作简便,具有重要的应用价值。     

The use of the nonradioactive digoxigenin chemiluminescent technology for plant genomic Southern blot hybridization: A comparison with radioactivity

A nonradioactive labelling and detection method for plant genomic DNA analysis is compared to the radioactive method. The radioisotopes are replaced by a nucleotide, digoxigenin-11-dUTP, and the signal detection is accomplished by the enzymatic reaction of alkaline phosphatase, conjugated to anti-digoxigenin antibodies, with the chemiluminescent substrate AMPPD (3-(2-spiroadamantane)-4-methoxy-4(3 phosphorytoxy) phenyl-1, 2-dioxetane). The sensitivity of the radioactive and nonradioactive methods are directly compared using identical Southern blots subjected to the radioactive and nonradioactive detection. The advantages of this nonradioactive method are discussed.     

吗啡耐受和电针耐受时大鼠脑内血管紧张素原的基因表达加速

我们以往的工作表明,脑内血管紧张素Ⅱ(AⅡ)作为一种抗阿片物质参与吗啡耐受和电针耐受。本工作探讨在此过程中脑内AⅡ的基因表达是否加速。采用酚抽提法提取大鼠脑组织总RNA,使用人工合成的寡脱氧核糖核酸探针进行打点杂交。放射自显影结果表明,多次皮下注射吗啡或连续数小时电针的大鼠脑血管紧张素原(Ang)mRNA含量明显升高。在IBAS图象分析仪上,测量各杂交点自显影曝光斑的积分光密度值(I.O.D.)表明,连续注射吗啡1d或4d使Ang mRNA分别增高1倍或8倍;电针3或6h使Ang mRNA分别增高5倍或13倍。说明大鼠经3—4h吗啡或电针处理,即可引起在转录水平上脑内Ang基因表达加强。