Detection of Viral DNA Sequences in Adenovirus-Transformed Cells by In Situ Hybridization

Cytological preparations of cells transformed by members of three groups of human adenoviruses, adenovirus 12, 7, and 2, were annealed with radioactive complementary RNA (cRNA) (4 x 10(7) to 4.5 x 10(7) dpm/mug) prepared by copying viral DNA with the Escherichia coli DNA-directed RNA polymerase. These in situ hybridizations detected adenovirus-specific DNA sequences in interphase nuclei when transformed cells were annealed with homologous viral cRNA, but not with heterologous viral cRNA. The highest autoradiographic grain counts were found over adenovirus 7-transformed cell nuclei, next over adenovirus 12-, and the lowest over adenovirus 2-transformed cell nuclei. This is the same order as found by reassociation kinetic measurements (K. Fujinaga and M. Green, unpublished data).     

PCR—微孔板杂交法检测不同人群TTV DNA

根据报道的TTV全序列设计引物和探针,建立PCR－微孔板杂交法,检测81例正常人群、92例职业献血员123例甲－庚型肝炎、32例非甲－庚型肝炎、48型发性肝癌患者的TTV DNA。结果表明TTV在以上五种人群中的阳性率分别为3．7％、4．3％、21．1％、28．1％、52．0％,前者与后三者比较有显著性差异（P＜0．05）,TTV合并HBV二重感染重叠感染的54．0％,这揭示不同人群均存在TTV感染,正常人群和职业献血员存在健康携带状态,甲－庚型肝炎和非甲－庚肝炎病人为高危人群,TTV可与各型肝炎存在重叠感染,TTV除经血传播外,存在其它传播途径,TTV感染与ALT及TBIL的升高密切相关。     

PCR-微孔板杂交法检测不同人群TTV DNA

根据报道的TTV全序列设计引物和探针,建立PCR-微孔板杂交法,检测81例正常人群、92例职业献血员123例甲～庚型肝炎、32例非甲～庚型肝炎、48例原发性肝癌患者的TTVDNA.结果表明TTV在以上五种人群中的阳性率分别为3.7%、4.3%、21.1%、28.1%、52.0%,前者与后三者比较有显著性差异(P＜0.05),TTV合并HBV二重感染占重叠感染的54.0%.这揭示不同人群均存在TTV感染,正常人群和职业献血员存在健康携带状态,甲～庚型肝炎和非甲～庚肝炎病人为高危人群,TTV可与各型肝炎存在重叠感染,TTV除经血传播外,存在其它传播途径,TTV感染与ALT及TBIL的升高密切相关.     

Reliability of the RNA-DNA Filter Hybridization for the Detection of Oncornavirus-Specific DNA Sequences

Denatured DNA from leukemic myeloblasts or uninfected chicken embryos, immobilized on nitrocellulose filters, was hybridized to a vast excess of [(3)H]70S RNA from purified avian myeloblastosis virus. The viral RNA was eluted from the RNA-DNA hybrids, purified, and then rehybridized in solution to an excess of either leukemic or normal chicken embryonic DNA. This study revealed that all the slow and the fast hybridizing viral RNA sequences detectable by liquid hybridization in DNA excess had hybridized to the filter bound DNA. Both techniques also gave similar values for the number of 28S ribosomal RNA genes contained in a chicken cell genome: 210 by the liquid hybridization procedure and 218 by the filter hybridization technique. Therefore, filter hybridization can accurately detect DNA sequences present in relatively few numbers in the genome of higher organisms.     

Isolation of Rhizobium loti Strain-Specific DNA Sequences by Subtraction Hybridization

Mixed-phase (heterogeneous) and single-phase (homogeneous) DNA subtraction-hybridization methods were used to isolate specific DNA probes for closely related Rhizobium loti strains. In the heterogeneous method, DNA from the prospective probe strain was repeatedly hybridized to a mixture of DNA from cross-hybridizing strains (subtracter DNA) which was immobilized on an epoxy-activated cellulose matrix. Probe strain sequences which shared homology with the matrix-bound subtracter DNA hybridized to it, leaving unique probe strain sequences in the mobile phase. In the homogeneous method, probe strain sequences were hybridized in solution to biotinylated, mercurated subtracter DNA. Biotinylated, mercurated subtracer DNA and probe strain sequences hybridized to it were removed by two-step affinity chromatography on streptavidin-agarose and thiol-Sepharose. The specificity of the sequences remaining after subtraction hybridization by both methods was assessed and compared by colony hybridization with R. loti strains. Both methods allowed the rapid isolation of strain-specific DNA fragments which were suitable for use as probes.     

Detection of a Novel Sepiapterin Reductase mRNA: Assay of mRNA in Various Cells and Tissues of Various Species

Fragments of cDNA coding for rat, murine, and human sepiapterin reductase (SR) were amplified by PCR via primer positioning close to the reported 3′-end of the coding region in the rat enzyme. They were sequenced and used as probes for mRNA detection. Northern blot analysis detected two mRNA species for SR. Their sizes were 1.3 and 2.1 kb for rat, 1.3 and 2.3 kb for mouse, and 1.6 and 2.1 kb for human cell lines. Comparison of rat cell lines and rat tissues indicated that in tissues only the 1.3-kb species is present. Washing of the Northern blots under different stringency conditions indicated a more stable interaction of the 1.3-kb mRNA species with the cDNA probe as compared to the 2.3-kb species. The 1.3-kb species corresponds to the reported 28.2-kDa molecular mass of rat SR monomer. SR mRNA expression is absent in the human NK-like cell line YT and in the murine erythroleukemia subclone B8/3, which both lack SR activity. Moreover, the relative mRNA expression correlates with the enzymatic activities of different cell lines within the same species. This indicates that SR activity is regulated by its steady state mRNA levels.     

哺乳动物凋亡相关基因p53同源序列在玉米(Zea mays L.)中的检出及其染色体原位杂交定位(英文)

p53基因普遍存在于动物组织中,是一个高度保守的肿瘤抑制基因,对细胞的生长、增殖和分化等多种发育程序进行调控。p53基因也是一个重要的细胞凋亡相关基因,决定着多种动物细胞的凋亡。有报导：用人P53抗体和p53基因的cDNA探针在玉米（ZeamaysL．）中检出了P53的同源蛋白及相应的mRNA,并初步确定其在功能上与动物中的P53蛋白非常相似。本实验首先用人p53基因的cDNA为探针,经SouthernBlotting初步确定其同源序列的存在（Fig．1）,然后进一步用生物素标记的原位杂交（DAB－ISH）和荧光原位杂交（FISH）对这些同源序列进行了染色体定位。DAB－ISH（Plate1）和FISH（Plate2）得到了一致的结果,在5S（第5染色体短臂）次末端、IL（第1染色体长臂）近末端、8L中部、3L中部近着丝粒以及9L近中部均镜检到p53基因探针的杂交信号,信号与着丝粒的百分距离分别为70．0±3．2、89．1±1．3、50．5±1.1、37．0±0．3和66．7±2．0（Tab．1,Fig．2）。利用异源探针是寻找植物凋亡相关基因的一种重要手段,也是目前国外的研究热点之一。本研究首次从DNA水平上证明了p53基因在玉米中的存在。为寻找和研究植物细胞凋亡基因提供了重要线索。     

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。     

Detection and Fluorescence in Situ Hybridization of Homologous Sequences for Mammalian Gene rb Related to Apoptosis in Maize

Human gene rb related to apoptosis was used as the probe for the Southern blot hybridization of the genomic DNA in both maize ( Zea mays L. ) and rice ( Oryza sativa L. ). The results indicated that the homologous sequences of rb were presented in the two species. The physical location of the rb homologous sequences was also carried out in maize chromosomes by fluorescence in sim hybridization (FISH). The gene rb was hybridized onto the long arms of the chromosomes 5 and 6, and the short ann of the chromosome 8. The detection rates of FISH were 7.58%, 16.16% and 10.10%, and percent distances from centromere to the detection sites were 86.17 + 3.22, 94.10 + 2.59 and 92.47 + 2.33 respectively. These results provided important clues to further research of plant apoptosis genes.     

玉米中哺乳动物细胞凋亡相关基因rb同源序列的检出及其荧光原位杂交

细胞凋亡 (ａｐｏｐｔｏｓｉｓ)是当今发育生物学研究的热点之一。成视网膜细胞瘤 (ｒｅｔｉｎｏｂｌａｓｔｏｍａ ,ｒｂ)基因是动物中第一个被克隆的肿瘤抑制基因 ,当细胞中缺失ｒｂ产物时 ,导致细胞凋亡。本研究采用Ｓｏｕｔｈ ｅｒｎ杂交技术 ,进一步证明玉米和水稻基因组中具有ｒｂ基因的同源序列 ,并利用染色体原位杂交技术 ,首次确定了这一序列在玉米染色体上的位置。1　材料和方法1 1　实验材料供试玉米 (ＺｅａｍａｙｓＬ .)品种为“黄早四”和“大红袍” ,供试水稻 (ＯｒｙｚａｓａｔｉｖａＬ .)品种为“鄂早 6号”。人ｒｂ基因的…     

Identification by Subtractive Hybridization of Sequences Specific for Salmonella enterica Serovar Enteritidis

Salmonella enterica serovar Enteritidis, a major cause of food poisoning, can be transmitted to humans through intact chicken eggs when the contents have not been thoroughly cooked. Infection in chickens is asymptomatic; therefore, simple, sensitive, and specific detection methods are crucial for efforts to limit human exposure. Suppression subtractive hybridization was used to isolate DNA restriction fragments present in Salmonella serovar Enteritidis but absent in other bacteria found in poultry environments. Oligonucleotide primers to candidate regions were used in polymerase chain reactions to test 73 non-Enteritidis S. enterica isolates comprising 34 different serovars, including Dublin and Pullorum, two very close relatives of Enteritidis. A primer pair to one Salmonella difference fragment (termed Sdf I) clearly distinguished serovar Enteritidis from all other serovars tested, while two other primer pairs only identified a few non-Enteritidis strains. These primer pairs were also useful for the detection of a diverse collection of clinical and environmental Salmonella serovar Enteritidis isolates. In addition, five bacterial genera commonly found with Salmonella serovar Enteritidis were not detected. By treating total DNA with an exonuclease that degrades sheared chromosomal DNA but not intact circular plasmid DNA, it was shown that Sdf I is located on the chromosome. The Sdf I primers were used to screen a Salmonella serovar Enteritidis genomic library and a unique 4,060-bp region was defined. These results provide a basis for developing a rapid, sensitive, and highly specific detection system for Salmonella serovar Enteritidis and provide sequence information that may be relevant to the unique characteristics of this serovar.     

荧光素标记的庚型肝炎病毒基因探针的制备及应用

The sequences of HGV gene that had been reported were checked and analyzed. After comparison of HGV seqences from different virus strains, a gene fragment of 31 nucleotides (7121-7152 nt) served as a probe was labeled by fluorescein-N₆-dd ATP with terminal transferase. The specificity of HGV probe was tested by dot-blot hybridization with HCV RNA—related virus RNA, C.T DNA, HGV-RNA, and Southern-blot hybridization with RT-PCR product of HGV. Positive results obstained only from the HGV-RNA of positive sera that was confirmed by nested RT-PCR, all others were negative. The sensitivity of fluorescein-labeled probe was examined. The result showed that the fluorescein-labeled probe was able to check≥1.56 pg of target genome. The conformity of HGV-RNA detection using RT-PCR and HGV gene probe was 97.9%. The experiment suggested that fluorescein-labeled probe can be used in clinic detection and epidemic study.     

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 µm tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.     

Detection of Low-Copy-Number Genomic DNA Sequences in Individual Bacterial Cells by Using Peptide Nucleic Acid-Assisted Rolling-Circle Amplification and Fluorescence In Situ Hybridization

An approach is proposed for in situ detection of short signature DNA sequences present in single copies per bacterial genome. The site is locally opened by peptide nucleic acids, and a circular oligonucleotide is assembled. The amplicon generated by rolling circle amplification is detected by hybridization with fluorescently labeled decorator probes.     

Molecular Modeling Study on the Enantioselective Binding of S- and R-Ofloxacin to Various DNA Sequences

Abstract

The complex formation of S- and R-ofloxacin with the self-complementary oligonucleotides, namely d[ATAGCGCTAT]₂, d[GCGATATCGC]₂ and d[ATAICICTAT]₂, were investigated by the molecular dynamics (MD) simulation. Four starting positions, including two intercalation positions with different insertion directions and two minor groove binding positions, were considered. The total energy of both S- and R-ofloxacin-d[ATAGCGCTAT]₂ complex, in which ofloxacin binds in the minor groove of the oligonucleotide, were lower than any intercalation binding mode. For both enantiomers, formation of the complex with GC oligonucleotide is more favorable than AT and IC oligonucleotides. When S- and R-ofloxacin are compared, the S-enantiomer exhibits more favorable total energy and torsion angles in the complex formation. This result is in agreement with the experimental observation [Hwangbo et al., Eur J Pharm Sci 18, 197 (2003)]. In the complex, both enantiomers form two hydrogen bonds: one between the carbonyl group of ofloxacin and the amine group of G16 and the other between the fluorine group and the G6 amine for S-ofloxacin. However, only one hydrogen bond is formed between endocyclic hydrogen atom at the C2 position of adenine and inosine base and carbonyl group of ofloxacin, which may be the reason for the GC preferentiality of ofloxacin.     

液相杂交快速检测特异DNA

检测特异DNA片段的方法中,传统Southern blot技术由于其高度可重复性及能够显示条带大小的特性,一直是DNA检测的“黄金标准”.但是杂交时间长,步骤复杂,放射性污染等问题亟待解决.为了简化Southern blot,研究使用了一种液相杂交快速检测DNA的方法,即使用异硫氰酸荧光素(FITC)标记的dUTP掺入探针后,在溶液中与待检测DNA样本42℃下杂交,然后琼脂糖凝胶电泳检测荧光杂交信号.利用质粒为模板,优化了探针制作、杂交液组成、杂交时间和温度等参数.在FITC-dUTP∶ dTTP比例为1∶3、模板质粒浓度为50μg、1×杂交缓冲液(25 mmol/LTris,10mmol/L EDTA,8mmol/L Nacl,PH =8.0)中95℃变性5～9 min和42℃杂交3h的实验条件下,可检出1.2μg的质粒,探针灵敏度为7.3 ng/μl.这种方法不需要转膜,曝光,大大节约了时间,简化了操作,荧光检测也为该方法同时检测多色样本提供了可能,可广泛应用于核酸检测.