The discrimination of high-risk HPV types by in situ hybridization and the polymerase chain reaction

Summary The parameter Tm^t has been defined by non-isotopic in situ hybridization and describes the tissue melting temperature (Tm^t) of human papillomavirus (HPV) DNA sequences. In this study, multiple in situ hybridization signals for HPV types 16, 31 and 33 in individual archival biopsies hybridized with genomic probes are shown by polymerase chain reactions to be due to cross-hybridization of probe sequences to a single tissue target. Tm^t is independent of viral type but depends on the homology between probe and target when using nick-translated whole genomic probes. The difference between Tm and Tm^t is not due to the presence of viral capsid protein. Multiple HPV signals in archival material should not therefore be interpreted as indicative of multiple HPV infection unless adequate stringency conditions have been employed or they are present in morphologically distinct areas of the biopsy.Furthermore, extrapolation of calculated DNA homologies to non-isotopic in situ hybridization analysis may not be appropriate. A hybridization signal does not imply probe and target identity: this has implications for HPV typing in clinical material.     

Discrimination of closely homologous HPV types by nonisotopicin situ hybridization: definition and derivation of tissue melting temperatures

Summary It is generally assumed that nucleic acid association duringin situ hybridization reactions is similar to that of nucleic acid association in solution. This assumption has been investigated by detecting closely homologous human papillomavirus types 6 and 11 byin situ hybridization as a model for the evaluation of stringency conditions in clinical biopsies.By examining matched and mismatched, labelled and target sequences under various stringency conditions, empirical DNA-DNA stability curves and their derivative equations for tissue melting temperatures (Tm^t) were derived. The corresponding values for Tm^t are 10–20°C higher than their solution equivalents. These data, supported by polymerase chain reaction experiments, demonstrate that closely homologous viral DNAs cross linked in tissue by formaldehyde fixation do not interact with the corresponding labelled probes as predicted from solution kinetic equations. This not only has theoretical implications but is also relevant to the accuracy of clinical diagnostic testing.     

In situ localization of PCR-amplified DNA and cDNA

Combining the high sensitivity of PCR with the cell localizing ability ofin situ hybridization allows for the reproducible detection of low copy targets in intact cells. This article describes several key variables that include fixation, protease digestion, the hot start maneuver, stringency, and, for RNA analysis, DNase digestion that are important to successfulin situ PCR. Also stressed is the importance of performing and interpreting controls with each experiment. Important controls include omission of key components, use of samples known either to contain or lack the target of interest and, most importantly, the in-built controls invariably present in the heterogeneous component of any given tissue type.     

Detection and localization onDrosophila melanogaster polytene chromosomes of sequences homologous to oncogeneyes

Sequences homologous to oncogeneyes (Y73/Esh/sarcoma viral oncogene cDNA) in theDrosophila melanogaster Oregon genome were detected byin situ hybridization on salivary gland chromosomes. Three separate sites, 8D/X, 57BC/2R and 95CD/3R, were identified. Presence of sequences highly homologous toyes in the genomic DNA was confirmed by dot blot hybridization under high stringency conditions.     

基因组原位杂交技术及其在园艺植物基因组研究中的应用

基因组原位杂交(GISH)技术可以鉴定植物多倍体物种起源、杂种亲本染色体来源和组成,分析栽培种与其近缘野生种的亲缘关系,研究减数分裂染色体行为等。基因组原位杂交包括多色基因组原位杂交、比较基因组原位杂交和自身基因组原位杂交等。基因组原位杂交技术的关键步骤是染色体制片、探针制备及长度优化、探针与封阻的浓度比例和杂交后洗脱强度。该文对近年来国内外有关基因组原位杂交技术的发展及其在园艺植物基因组研究中的应用现状进行了综述,并指出随着多种园艺植物全基因组的测定,未来应从基因组信息中寻找更多的染色体特异性标记,结合荧光显带及荧光原位杂交技术,为深入研究园艺植物的起源以及遗传关系鉴定等提供技术支持。     

Mg2+ ions bind at the C‐terminal region of skeletal muscle α‐tropomyosin

Tropomyosin (Tm) is a dimeric coiled‐coil protein that polymerizes through head‐to‐tail interactions. These polymers bind along actin filaments and play an important role in the regulation of muscle contraction. Analysis of its primary structure shows that Tm is rich in acidic residues, which are clustered along the molecule and may form sites for divalent cation binding. In a previous study, we showed that the Mg²⁺‐induced increase in stability of the C‐terminal half of Tm is sensitive to mutations near the C‐terminus. In the present report, we study the interaction between Mg²⁺ and full‐length Tm and smaller fragments corresponding to the last 65 and 26 Tm residues. Although the smaller Tm peptide (Tm_{259‐284(W269)}) is flexible and to large extent unstructured, the larger Tm_{220‐284(W269)} fragment forms a coiled coil in solution whose stability increases significantly in the presence of Mg²⁺. NMR analysis shows that Mg²⁺ induces chemical shift perturbations in both Tm_{220‐284(W269)} and Tm_{259‐284(W269)} in the vicinity of His276, in which are located several negatively charged residues. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 583–590, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Instability in the Central Region of Tropomyosin Modulates the Function of Its Overlapping Ends

The causal link between disparate tropomyosin (Tm) functions and the structural instability in Tm is unknown. To test the hypothesis that the structural instability in the central region of Tm modulates the function of the overlapping ends of contiguous Tm dimers, we used transgenic mice (Tm_DM) that expressed a mutant α-Tm in the heart; S229E and H276N substitutions induce structural instability in the central region and the overlapping ends of Tm, respectively. In addition, two mouse cardiac troponin T mutants (TnT_1–44Δ and TnT_45–74Δ) that have a divergent effect on the overlapping ends of Tm were employed. The S229E-induced instability in the central region of Tm_DM altered the overlapping ends of Tm_DM, thereby it negated the attenuating effect of H276N on Ca²⁺-activated maximal tension. The rate of cross-bridge detachment (g) decreased in Tm_DM+TnT_WT and Tm_H276N+TnT_WT fibers but increased in Tm_DM+TnT_45–74Δ fibers; however, TnT_45–74Δ did not alter g, demonstrating that S229E in Tm_DM had divergent effects on g. The S229E substitution in Tm_DM ablated the H276N-induced desensitization of myofilament Ca²⁺ sensitivity in Tm_DM+TnT_1–44Δ fibers. To our knowledge, novel findings from this study show that the structural instability in the central region of Tm modifies cardiac contractile function via its effect on the overlapping ends of contiguous Tm.     

Instability in the Central Region of Tropomyosin Modulates the Function of Its Overlapping Ends

The causal link between disparate tropomyosin (Tm) functions and the structural instability in Tm is unknown. To test the hypothesis that the structural instability in the central region of Tm modulates the function of the overlapping ends of contiguous Tm dimers, we used transgenic mice (Tm_DM) that expressed a mutant α-Tm in the heart; S229E and H276N substitutions induce structural instability in the central region and the overlapping ends of Tm, respectively. In addition, two mouse cardiac troponin T mutants (TnT_1–44Δ and TnT_45–74Δ) that have a divergent effect on the overlapping ends of Tm were employed. The S229E-induced instability in the central region of Tm_DM altered the overlapping ends of Tm_DM, thereby it negated the attenuating effect of H276N on Ca²⁺-activated maximal tension. The rate of cross-bridge detachment (g) decreased in Tm_DM+TnT_WT and Tm_H276N+TnT_WT fibers but increased in Tm_DM+TnT_45–74Δ fibers; however, TnT_45–74Δ did not alter g, demonstrating that S229E in Tm_DM had divergent effects on g. The S229E substitution in Tm_DM ablated the H276N-induced desensitization of myofilament Ca²⁺ sensitivity in Tm_DM+TnT_1–44Δ fibers. To our knowledge, novel findings from this study show that the structural instability in the central region of Tm modifies cardiac contractile function via its effect on the overlapping ends of contiguous Tm.     

Detection of nerve growth factor and its mRNA by separate and combined immunohistochemistry andin situ hybridization in mouse salivary glands

Summary Intense labelling of secretory cells in the male mouse submandibular gland was observed afterin situ hybridization using mouse nerve growth factor (NGF) cDNA probes. Under the same conditions, sparse less intensely labelled cells were also found in the sublingual gland. Hybridization to a chicken NGF cDNA probe gave weak labelling on the glands in accordance with a weak cross-hybridization between mouse NGF mRNA and chicken NGF cDNA probes, whereas no labelling was seen using pUC9 DNA as a hybridization probe. A combination ofin situ hybridization and immunohistochemistry was also carried out on the same sections of submandibular gland. A good correlation was seen between actively synthesizing and intensely immunoreactive cells in the gland. The technique described here allows the detection of individual cells synthesizing relatively low levels of NGF. The combination ofin situ hybridization and immunocytochemistry on the same section should be particularly useful in cases where NGF is transported away from its site of synthesis.     

Diel Division Cycle and Growth Rates of Synechococcus in Lakes Huron and Michigan1

A clone of Synechococcus isolated from Lake Huron and natural populations of Synechococcus from lakes Huron and Michigan were studied in 1989 to examine the diel division cycle and to provide estimates of the in situ growth rate based on the frequency of dividing cells (FDC) method. Cultured populations of Synechococcus exhibited a consistent diel division pattern with a midday/afternoon (1100–1800 h) peak in the percent of dividing cells. The maximum percent of dividing cells varied among cultures (8-27%) and was related to the growth rate. A small fraction of dividing cells (3-5%) remained throughout the dark period, suggesting that some cells were arrested in the doublet stage prior to division. The duration of division (t_d) ranged from 2.6-4.9 h, with a 3.7 h mean for cultures with growth rates ≥0.34 d⁻¹ but increased to 8 h at a lower growth rate of 0.20 d⁻¹. The diel division pattern for natural populations was very similar to the laboratory clone; an afternoon peak (1400-2100 h) in dividing cells and a small fraction of dividing cells (2-5%) remained during the dark period. The maximum percent of dividing cells for natural populations ranged from 6-10%. In situ growth rates, determined from the FDC and assuming a constant t_d of 3.7 h, ranged from 0.30-0.42 d⁻¹. The FDC method may provide accurate estimates of in situ growth, particularly in environments where the growth rate is >0.34 d⁻¹, but in lakes Huron and Michigan where growth rates can be lower and t_d values may increase, FDC-growth rates must be viewed with caution.     

The effects of varying key steps in the non-radioactive in situ hybridization protocol: a quantitative study

Summary In situ hybridization represents a major advance in the study of gene expression and, thus, in the evaluation of cellular function in histological sections. The availability of oligonucleotide probes labelled with biotin and sensitive immunohistochemical detection systems makes the study of different types of mRNA by in situ hybridization easier. However, a large number of protocols have been reported, which is sometimes confusing. The present study analyses quantitatively the influence of each important step of in situ hybridization on the staining intensity of rat proinsulin mRNA. The aim was to optimize technical conditions, to make the method sensitive and to evaluate its reproducibility for proinsulin mRNA detection and measurements. The duration of fixation and the digestion have an important impact on the results. The optimal digestion time depends on the fixation. With a digestion of 30 g ml^–1 proteinase K for 12 min at 37°C, the optimal fixation time was 24 h. Section thickness also influences the staining intensity. The intensity of the staining increases as the section thickness increases from 3 to 5 m before slowly decreasing. A weak paraformaldehyde post-fixation (0.4% for 20 min) gives best results in comparison to a stronger post-fixation (4% for 10 min). An increase of probe concentration leads to a higher specific labelling, reaching a plateau at 800 ng ml^–1. Hybridization temperature (37–42°C) exerts little influence. However, the temperature of the washes and the immunodetection system have a major effect on the intensity of labelling.Quantification has permitted the evaluation of the influence of each key for optimizing and standardizing the non-radioactive in situ hybridization protocol. In these well-defined conditions, the intra and inter-assay coefficient of variation remains lower than 6% and thus the method can be used to quantify the content of proinsulin mRNA or other specific mRNAs in experimental and pathological conditions.     

5-HT receptors in mammalian brain: receptor autoradiography andin situ hybridization studies of new ligands and newly identified receptors

Summary In recent years the family of mammalian serotonin receptors has grown to 14 different subtypes, characterized by pharmacological or molecular biological techniques. In parallel, new ligand molecules have been developed for their study. However, selective ligands are not yet available to study every one of them. In addition the degree of selectivity of ligands, hitherto regarded as specific for a particular receptor subtype has been called in question by their affinities for newly discovered receptors. Consequently, a re-evaluation of past ligand receptor autoradiography work is necessary in view of the redefined receptor profiles of these ligands, and the introduction of newly developed ligands. A further difficulty for the characterization of these receptors is the absence of selective antagonist ligands which, for some of the subtypes, have become available only recently. In an attempt to overcome these difficulties we have combinedin situ hybridization histochemistry and receptor ligand autoradiography to study the regional and cellular localization of several serotonin receptors in the rodent brain. In addition, for some receptors, we have expanded these studies to primates, including humans. We have found that the distribution of 5-HT_1A receptors in monkey brain, labelled with the agonist³H-8-OH-DPAT and the antagonist³H-WAY 100635 was very similar at the levels examined, and corresponded well with that observed for the cells containing mRNA coding for this receptor, confirming the somatodendritic localization of 5-HT_1A receptors in monkey brain. The labelling conditions to visualize 5-HT_1F receptors in guinea pig brain, namely³H-sumatriptan in the presence of 10⁻⁸ m 5-CT to block 5-HT_1D receptors, are suitable for visualizing this receptor, since the results agreed with those observed byin situ hybridization. By using³H-ketanserin and³H-mesulergine in parallel within situ hybridization using the corresponding oligonucleotides, we were able to show that these ligands label respectively 5-HT_2A and 5-HT_2C binding sites in monkey brain. 5-HT₄ receptors were localized in the brain of several species including humans by using¹²⁵I-SB 207710.In situ hybridization experiments performed in guinea pig confirmed that 5-HT₄ receptors are localized on the terminals of the striatopallidal and striatonigral projections. 5-HT₇ binding sites were labelled in rat and guinea pig brains by incubating with³H-5-CT in the presence of 100 μm WAY 100135 and 250 μm GR 127935; the distribution obtained in both species agreed, in general, with that of the corresponding mRNA coding for them. These results are an illustration of the understanding of our current knowledge of the chemical neuroanatomy of the mammalian 5-HT system.     

Improved procedure for the measurement of telomere length in whole cells by PNA probe and flow cytometry

Objectives: Peptide nucleic acid (PNA) probes hybridize to denatured telomeric sequences in cells permeabilized in hot formamide. In reported protocols, the hybridization was conducted in solutions with high formamide concentrations to avoid the DNA renaturation that can hamper binding of the oligo‐PNA probe to specific sequences. We postulated that telomeric DNA, confined in the nuclear microvolume, is not able to properly renature after hot formamide denaturation. Therefore, to improve hybridization conditions between the probe and the target sequences, it might be possible to add probe to sample after the complete removal of formamide. Materials and methods: After telomeric DNA denaturation in hot formamide solution and several washes to remove the ionic solvent, cells were hybridized overnight at room temperature with human telomere‐specific PNA probe conjugated with Cy5 fluorochrome, Cy5‐OO‐(CCCTAA)₃. After stringency washes and staining with ethidium bromide, the cells were analysed by flow cytometry and by using a confocal microscope. Results: Using three continuous cell lines, different in DNA content and telomere length, and resting human peripheral blood T and B lymphocytes, we demonstrated that the oligo‐PNA probe hybridized to telomeric sequences after complete removal of formamide and that in the preserved nucleus, telomeric sequence denaturation is irreversible. Conclusion: According to our experience, oligo‐PNA binding results is efficient, specific and proportional to telomere length. These, our original findings, can form the technological basis of actual in situ hybridization on preserved whole cells.     

Species-specific FISH analysis of cecal microflora in rats administered with lactic acid bacteria

Summary We investigated the effects of lactic acid bacterial (LAB) cells in rats using fluorescence in situ hybridization (FISH) targeting 16S rRNA to identify the cecal microbial community. We designed a novel species-specific 16S rDNA probe to detect Lactobacillus rhamnosus (Lrham454). Subtractive technique using the LAC722 probe (Sakai et al. 2004 Journal of Bioscience and Bioengineering 98, 48) under different hybridization stringency (LAC722(L-H)) was applied to identify Lactococcus lactis. We also applied Lplan447 and LAC722(L) to detect Lactobacillus plantarum and a wide range of LAB (total LAB), respectively. We optimized the hybridization and washing conditions and then quantified L. rhamnosus, L. plantarum, and L. lactis cells in rat cecal contents. We monitored increases in individual bacterial populations and in total LAB caused by the administration of the corresponding LAB cells. Growth, food efficiency and internal disorders did not significantly differ among the rats administered with LABs. Rats administered with polydextrose (POL) developed diarrhea, which decreased the total numbers of cecal bacteria, whereas the simultaneous administration of POL and L. rhamnosus KY-3 eased this symptom, and recovered the numbers of total LAB and of L. rhamnosus.     

Characteristics of a phylogenetically ambiguous,arsenic-oxidizing <Emphasis Type="Italic">Thiomonas</Emphasis> sp., <Emphasis Type="Italic">Thiomonas arsenitoxydans</Emphasis> strain 3As<Superscript>T</Superscript> sp. nov

A moderately acidophilic, facultative chemoautotrophic, As(III)-oxidizing Thiomonas sp. (strain 3As^T) was previously shown, on the basis of comparative 16S rRNA gene sequences, to be closely related to both Tm. perometabolis DSM 18570^T and Tm. intermedia DSM 18155^T. While it had shared many physiological traits with Tm. intermedia ^T, a mean DNA–DNA hybridization value (DDHV) of 47.2% confirmed that strain 3As^T was not a strain of Tm. intermedia, though the situation with regard to Tm. perometabolis (DDHV previously determined as 72%) was more ambiguous. A comparative physiological and chemotaxonomic study of strain 3As^T and Tm. perometabolis ^T was therefore carried out, together with multilocus sequence analysis (MLSA) of all three bacteria. Differences in fatty acid profiles and utilization of organic substrates supported the view that strain 3As^T and Tm. perometabolis are distinct species, while MLSA showed a closer relationship between strain 3As^T and Tm. intermedia ^T than between strain 3As^T and Tm. perometabolis ^T. These apparent contradictory conclusions were explained by differences in genome of these three bacteria, which are known to be highly flexible in Thiomonas spp. A novel species designation Thiomonas arsenitoxydans is proposed for strain 3As^T (DSM 22701^T, CIP 110005^T), which is nominated as the type strain of this species.     

Na2CO3 Influence on DNA Double Helix Stability: Strong Anion Destabilizing Effect

Abstract

Addition of Na₂CO₃ to almost salt-free DNA solution (5·10^?5M EDTA, pH=5.7, Tm=26.5 °C) elevates both pH and the DNA melting temperature (Tm) if Na₂CO₃ concentration is less than 0.004M. For 0.004M Na₂CO₃, Tm=58 °C is maximal and pH=10.56. Further increase in concentration gives rise to a monotonous decrease in Tm to 37 °C for 1M N₂CO₃ (pH=10.57). Increase in pH is also not monotonous. The highest pH=10.87 is reached at 0.04M Na₂CO₃ (Tm=48.3 °C). To reveal the cause of this DNA destabilization, which happens in a narrow pH interval (10.56÷10.87) and a wide Na₂CO₃ concentration interval (0.004÷1M), a procedure has been developed for determining the separate influences on Tm of Na⁺, pH, and anions formed by Na₂CO₃ (HCO₃ ^? and CO₃ ^2-). Comparison of influence of anions formed by Na₂CO₃ on DNA stability with Cl^? (anion inert to DNA stability), ClO₄ ^? (strong DNA destabilizing “chaotropic” anion) and OH^? has been carried out. It has been shown that only Na⁺ and pH influence Tm in Na₂CO₃ solution at concentrations lower than 0.001M. However, the Tm decrease with concentration for [Na₂CO₃]≥0.004M is only partly caused by high pH≈10.7. Na₂CO₃ anions also exert a strong destabilizing influence at these concentrations. For 0.1M Na₂CO₃ (pH=10.84, [Na⁺]=0.2M, Tm=42.7 °C), the anion destabilizing effect is higher 20 °C. For NaClO₄ (ClO₄ ^? is a strong “chaotropic” anion), an equal anion effect occurs at much higher concentrations ～3M. This means that Na₂CO₃ gives rise to a much stronger anion effect than other salts. The effect is pH dependent. It decreases fivefold at neutral pH after addition of HCl to 0.1M Na₂CO₃ as well as after addition of NaOH for pH>11.2.     

Probing the Flexibility of Tropomyosin and Its Binding to Filamentous Actin Using Molecular Dynamics Simulations

Tropomyosin (Tm) is a coiled-coil protein that binds to filamentous actin (F-actin) and regulates its interactions with actin-binding proteins like myosin by moving between three positions on F-actin (the blocked, closed, and open positions). To elucidate the molecular details of Tm flexibility in relation to its binding to F-actin, we conducted extensive molecular dynamics simulations for both Tm alone and Tm-F-actin complex in the presence of explicit solvent (total simulation time >400 ns). Based on the simulations, we systematically analyzed the local flexibility of the Tm coiled coil using multiple parameters. We found a good correlation between the regions with high local flexibility and a number of destabilizing regions in Tm, including six clusters of core alanines. Despite the stabilization by F-actin binding, the distribution of local flexibility in Tm is largely unchanged in the absence and presence of F-actin. Our simulations showed variable fluctuations of individual Tm periods from the closed position toward the open position. In addition, we performed Tm-F-actin binding calculations based on the simulation trajectories, which support the importance of Tm flexibility to Tm-F-actin binding. We identified key residues of Tm involved in its dynamic interactions with F-actin, many of which have been found in recent mutational studies to be functionally important, and the rest of which will make promising targets for future mutational experiments.     

Study of stringency conditions for human papillomavirus DNA detection on cell lines, frozen and paraffin-embedded tissue sections by in situ hybridization with biotinylated probes

In situ hybridization was mainly used for typing human papillomavirus (HPV) in paraffin-embedded or frozen sections under stringent conditions (SC). We tested 5 different conditions of stringency with biotinylated HPV 1, 2, 16 and 18 probes on 3 cell lines (Sihà and CaSki with HPV16, HeLa with HPV18) by varying the concentration of formamide in the hybridization mixture and washings in order to determine the stringency conditions to be used to assess the presence of HPV and its typing: A-low stringency, hybridization at 35 degrees C below the melting temperature of DNA (Tm-35 degrees C) and washings without formamide; B-low stringency, hybridization and washings at Tm-35 degrees C; C-medium stringency, hybridization at Tm-35 degrees C and washings at Tm-12 degrees C; D-high stringency, hybridization at Tm-12 degrees C and washing without formamide; E-very high stringency, hybridization and washings at -12 degrees C. This study showed that HPV typing required a high stringency. On the contrary, under non stringent conditions (NSC), each cell line was positive with the heterologous probes. When 3 to 5 stringency conditions were assayed on 4 frozen samples, similar results were obtained. Typing required high stringency conditions whereas NSC allowed HPV detection. Furthermore, this study demonstrated the specificity of the reaction in lesions positive with more than one type. Stringent (Tm-12 degrees C) and non stringent (Tm-35 degrees C) conditions of hybridization were further applied to 57 biopsy sections (17 frozen and 40 paraffin-embedded specimens) from typical wart lesions and lesions suspected of HPV.(ABSTRACT TRUNCATED AT 250 WORDS)