Monitoring of Ralstonia eutropha KT1 in groundwater in an experimental bioaugmentation field by in situ PCR.

Ralstonia eutropha KT1, which degrades trichloroethylene, was injected into the aquifer after activation with toluene, and then the number of bacteria was monitored by in situ PCR targeting the phenol hydroxylase gene and by fluorescent in situ hybridization (FISH) targeting 16S rRNA. Before injection of the bacterial suspension, the total concentration of bacteria in the groundwater was approximately 3 x 10(5) cells/ml and the amount of Ralstonia and bacteria carrying the phenol hydroxylase gene as a percentage of total bacterial cells was less than 0.1%. The concentration of bacteria carrying the phenol hydroxylase gene detected by in situ PCR was approximately 3 x 10(7) cells/ml 1 h after injection, and the concentration of Ralstonia detected by FISH was similar. The number of bacteria detected by in situ PCR was similar to that detected by FISH 4 days after the start of the extraction of groundwater. On and after day 7, however, the number of bacterial cells detected by FISH was less than that detected by in situ PCR.     

Visualization and enumeration of bacteria carrying a specific gene sequence by in situ rolling circle amplification

Rolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count single Escherichia coli cells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes inside E. coli cells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA. E. coli cells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 10(6)-fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria.     

Quantitative determination of free-DNA uptake in river bacteria at the single-cell level by in situ rolling-circle amplification

Detection of plasmid DNA uptake in river bacteria at the single-cell level was carried out by rolling-circle amplification (RCA). Uptake of a plasmid containing the green fluorescent protein gene (gfp) by indigenous bacteria from two rivers in Osaka, Japan, was monitored for 506 h using this in situ gene amplification technique with optimized cell permeabilization conditions. Plasmid uptake determined by in situ RCA was compared to direct counts of cells expressing gfp under fluorescence microscopy to examine differences in detection sensitivities between the two methods. Detection of DNA uptake as monitored by in situ RCA was 20 times higher at maximum than that by direct counting of gfp-expressing cells. In situ RCA could detect bacteria taking up the plasmid in several samples in which no gfp-expressing cells were apparent, indicating that in situ gene amplification techniques can be used to determine accurate rates of extracellular DNA uptake by indigenous bacteria in aquatic environments.     

Regional assignment of nonspecific cross-reacting antigen (NCA) of the CEA gene family to chromosome 19 at band q13.2

Nonspecific cross-reacting antigen (NCA) is a member of the carcinoembryonic antigen (CEA) gene family. Recently, a DNA segment for part of the human NCA gene was isolated and sequenced. We mapped this gene by Southern blot analysis of hybrid cells and by in situ hybridization. The Southern blot analysis indicated that the NCA gene is on human chromosome 19 and the in situ hybridizations localized the gene to band 19q13.2.     

Detection of the Leghemoglobin Gene on Two Chromosomes of Phaseolus vulgaris by in situ PCR Linked-Fluorescent in situ Hybridization (FISH)

The leghemoglobin (Lb) gene on the metaphase chromosomes ofPhaseolus vulgaris was amplified by in situ PCR. The amplifiedLb gene could be detected on two chromosomes by fluorescentin situ hybridization (FISH) using the short Lb gene probe. (Received January 9, 1998; Accepted April 30, 1998)     

Quantitative Determination of Free-DNA Uptake in River Bacteria at the Single-Cell Level by In Situ Rolling-Circle Amplification

Detection of plasmid DNA uptake in river bacteria at the single-cell level was carried out by rolling-circle amplification (RCA). Uptake of a plasmid containing the green fluorescent protein gene (gfp) by indigenous bacteria from two rivers in Osaka, Japan, was monitored for 506 h using this in situ gene amplification technique with optimized cell permeabilization conditions. Plasmid uptake determined by in situ RCA was compared to direct counts of cells expressing gfp under fluorescence microscopy to examine differences in detection sensitivities between the two methods. Detection of DNA uptake as monitored by in situ RCA was 20 times higher at maximum than that by direct counting of gfp-expressing cells. In situ RCA could detect bacteria taking up the plasmid in several samples in which no gfp-expressing cells were apparent, indicating that in situ gene amplification techniques can be used to determine accurate rates of extracellular DNA uptake by indigenous bacteria in aquatic environments.     

Monitoring of Ralstonia eutropha KT1 in Groundwater in an Experimental Bioaugmentation Field by In Situ PCR

Ralstonia eutropha KT1, which degrades trichloroethylene, was injected into the aquifer after activation with toluene, and then the number of bacteria was monitored by in situ PCR targeting the phenol hydroxylase gene and by fluorescent in situ hybridization (FISH) targeting 16S rRNA. Before injection of the bacterial suspension, the total concentration of bacteria in the groundwater was approximately 3 × 10⁵ cells/ml and the amount of Ralstonia and bacteria carrying the phenol hydroxylase gene as a percentage of total bacterial cells was less than 0.1%. The concentration of bacteria carrying the phenol hydroxylase gene detected by in situ PCR was approximately 3 × 10⁷ cells/ml 1 h after injection, and the concentration of Ralstonia detected by FISH was similar. The number of bacteria detected by in situ PCR was similar to that detected by FISH 4 days after the start of the extraction of groundwater. On and after day 7, however, the number of bacterial cells detected by FISH was less than that detected by in situ PCR.     

Visualization and Enumeration of Bacteria Carrying a Specific Gene Sequence by In Situ Rolling Circle Amplification

Rolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count single Escherichia coli cells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes inside E. coli cells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA. E. coli cells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 10⁶-fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria.     

nif HDK基因在W80-2上的初步定位

对几种质粒检测方法进行了比较 ,发现原位裂解法能比较满意地检测到巴西固氮螺菌 (Azospirillumbrasilense)的巨大质粒。利用改进后的原位裂解法能比较稳定地检测到W 80 2菌株中的巨大质粒。通过Southern blotting的方法将W 80 2菌的染色体及巨大质粒转到尼龙膜上 ,与用地高辛标记的含nifHDK基因的 pSA30质粒杂交 ,发现W80 2菌株的nifHDK基因定位在染色体上。     

Localization of the human coproporphyrinogen oxidase gene to chromosome band 3q12

The human gene encoding coproporphyrinogen oxidase is the defective gene in hereditary coproporphyria. This gene was mapped to chromosome band 3q12 using fluorescent in situ hybridization. The chromosomal localization was confirmed by cosegregation of the human gene with chromosome 3 in a panel of human/rodent somatic hybrids.     

MYB基因在玉米雌穗发育中差异表达的研究

目的:研究MYB基因在玉米雌穗不同发育阶段的表达情况,为探讨其生物学功能提供相关线索。方法:用芯片杂交的方法检测玉米雌穗早期发育过程中差异表达的MYB基因,定量PCR验证差异基因的表达情况,原位杂交分析差异基因的组织器官表达。结果:一个MYB基因在雌穗发育到小花分化期时上调表达(芯片分析其表达差异倍数为1.8)。其表达的差异性得到了定量PCR的验证(定量PCR分析其差异表达倍数为4.3)。原位杂交分析发现该基因主要表达于小穗的生长锥顶部和小花雌雄蕊原基部位。结论:MYB基因对玉米雌穗早期发育起到一定作用。     

Localization of the gene encoding human Factor V to chromosome 1q21–25

The gene encoding human coagulation Factor V (FV), one of the cofactors in the blood clotting process, has been mapped to chromosome 1 by both Southern hybridization to DNA from human-hamster somatic cell hybrids and in situ hybridization. The whole plasmid pUC3A containing a 1.5-kb cDNA sequence for FV was 32P-labeled for Southern analysis and 3H-labeled for in situ hybridization to metaphase chromosomes. The results localized the FV gene to the region of 1q21-25.     

BEK, a receptor for multiple members of the fibroblast growth factor (FGF) family, maps to human chromosome 10q25.3----q26.

The gene for the fibroblast growth factor receptor BEK was assigned to human chromosome 10 by applying polymerase chain reaction techniques to DNAs from a panel of human x rodent somatic cell hybrids. The gene was further localized to 10q25.3----q26 by in situ hybridization.     

Localization of the human oestrogen receptor gene to chromosome 6q24----q27 by in situ hybridization

The oestrogen receptor gene (ER) was mapped by in situ hybridization. Using a human cDNA probe containing the coding sequence for the oestrogen receptor, the gene was localized to 6q24----q27.     

Mapping the beta NGF gene in situ to a microchromosome in chicken.

The chicken nerve growth factor (beta NGF) gene has been mapped by fluorescent in situ hybridization to a pair of microchromosomes, confirming previous reports of the existence of a single gene locus. A 39-kb genomic fragment cloned in a cosmid vector and including the 5' end of the beta NGF locus was biotinylated for nonradioactive detection of the gene. This report adds to the increasing evidence proving microchromosomal localization of highly conserved and biologically fundamental genes. The implications of such genes belonging to very small linkage groups for the transmission of alleles from generation to generation together with the relevance of nonisotopic in situ hybridization for avian gene mapping are considered.     

褐飞虱取食诱导的MAPK3基因在水稻叶鞘组织中的定位

利用Northern杂交技术,对促分裂原活化蛋白激酶基因（MAPK3,BPHiw103）进行了表达分析,同时,针对抗虫水稻B5植株接种褐飞虱若虫48h后的叶鞘组织切片进行了原位定位。Northern杂交结果表明,在褐飞虱取食后,MAPK3 mRNA整体表现为上调的特性。原位杂交显示,褐飞虱取食前,MAPK3在水稻叶的薄壁组织中大量表达;而取食后,在韧皮部表达明显增加,在薄壁组织表达则呈下降趋势。这一点在叶心组织切片中表现最为明显。这些结果说明,水稻在受褐飞虱若虫取食诱导和刺激后,MAPK3的表达在受伤部位急剧增加,推测MAPK3基因可能在水稻对褐飞虱的抗性反应中发挥作用。     

Strain-dependent expression of VH gene families

The tremendous diversity of the antibody specificity repertoire stems from the ability of each developing B cell to select one out of many possible variable, diversity, and joining gene segments by specific rearrangement of the DNA. The mechanism by which V region gene segments is selected is not known. Moreover, evidence for both random and nonrandom expression of VH genes in mature B cells has been presented previously. In this report, the technique of in situ hybridization is used to accurately measure at the single cell level VH gene family expression in LPS-induced cells from several strains. In this way, at least one-third of the B cells are stimulated and a large sampling of activated splenocytes from each strain analyzed. The use of in situ hybridization eliminates any potential biases resulting from transformation protocols. In addition, because all populations of cells are analyzed by both in situ hybridization and immunocytochemical staining with anti-IgM, the proportion of cells detected by in situ hybridization could be compared with the proportion of B cells, blasts, and plasma cells in the population. It was concluded from these comparisons that the cells being detected by in situ hybridization under the conditions described are plasmablasts and plasma cells. Therefore, an accurate measure of the functional and expressed VH gene repertoire could be made. The results clearly demonstrate strain-dependent variation in VH gene family expression, particularly VH 7183 and VH J558 with up to three-fold differences observed. Thus, either there is considerable strain variation in the number of functional VH gene family segments or the expression of VH genes is not entirely random.     

Visualization of the cbbL gene in healthy and starved chemoautotrophic nitrifying bacteria

Three in situ methods of visualizing the cbbL gene in intact cells of nitrifying bacteria at different physiological states (dormant and metabolically active) were compared after epifluorescence microscopy and image analysis. FISH alone showed the weakest signal intensity. Direct in situ PCR, incorporating labeled nucleotides, showed the greatest sensitivity but also the greatest background. The combination of unlabeled in situ PCR followed by FISH showed relatively high sensitivity, along with the lowest background and highest specificity. Although functional gene expression was not examined in this study, visualization of the potential for carbon fixation in heterogeneous cultures of nitrifying bacteria was demonstrated.