In situ hybridization localizes avocado sunblotch viroid on chloroplast thylakoid membranes and coconut cadang cadang viroid in the nucleus

Viroids, small single-stranded circular RNA molecules, are the smallest known infectious agents in Nature. The apparent inability of viroids to encode for proteins means that they must rely fully on host functions for their replication. The specific ultrastructural localization of viroids is fundamental to the determination of their replication strategies. In this paper the first in situ hybridization study to localize viroids within the cell at the electron microscope level is reported. Biotin-labelled RNA probes were used with subsequent detection by gold-labelled monoclonal anti-biotin antibodies to localize avocado sunblotch viroid and coconut cadang cadang viroid. Avocado sunblotch viroid was located in chloroplasts, mostly on the thylakoid membranes of cells from infected leaves of avocado (Persea americana). In contrast, coconut cadang cadang viroid was located in the nucleolus and nucleoplasm of cells of infected leaves of oil palm (Elaeis guineensis), with a higher concentration in the nucleolus. The results provide insight on the potential host RNA polymerases involved in the replication of these two viroids.     

Application of rapid biochemical methods for detecting avocado sunblotch disease

The method of Palukaitis & Symons (1980) for extracting low molecular weight ribonucleic acids from plant tissue was improved by CF-11-cellulose chromatography and further simplified for use in routine biochemical indexing for avocado sunblotch viroid (ASBV). Extracts were prepared routinely at 10 g dry weight equivalents (DWE) of tissue per ml; more concentrated than previously possible with many avocado cultivars. Conditions for assaying extracts of ASBV were standardised and the lower limits for detection determined as 80–230 ng ASBV/g DWE of tissue for polyacrylamide gel electrophoresis (PAGE) and 1 ng/g DWE of tissue for complementary DNA (cDNA) probe assays. The concentration of ASBV in a single infected tree varied from 5 to 5000 ng/g DWE between branches, but only to a minor degree between mature leaves and young blossoms within branches. Six independent sources of sunblotch disease were examined and all proved positive for ASBV by PAGE. The ASBV extracted from five of these sources hybridised with cDNA prepared from the sixth or standard source (Hass/SB-1), with hybridisation values ranging from 43% to 89%. In a survey of 76 trees intended for propagation in Australia, all of 17 trees previously accepted as healthy on the basis of graft transmission tests were negative for ASBV by PAGE and had cDNA hybridisation values ranging from 1.8% to 12.1%. Amongst 59 trees apparently free of sunblotch symptoms but not previously indexed, only one tree was positive for ASBV by both PAGE and cDNA probe assay. The other 58 trees were negative by PAGE but had hybridisation values ranging from 1.0% to 42.8%. Forty-nine trees had values consistent with known healthy trees (<12% hybridisation), while the results of the remaining nine trees will require confirmation by additional tests before a conclusion about ASBV is made. The cDNA probe assay successfully detected ASBV in avocado seedlings graft inoculated with Hass/SB-1, 1–2 months before symptoms were displayed but not until 6 months after inoculation. Methods for improving the cDNA probe assay still further are discussed.     

Detection of avocado sunblotch viroid (ASBV) by dot-spot self-hybridization with a [32P]-labelled ASBV-RNA

RNA was extracted from plants infected with avocado sunblotch viroid (ASBV) and was analyzed by electrophoresis in polyacrylamide gel. The ASBV related fraction was eluted from the gel, labelled with [³²P] using polynucleotide kinase and used as a probe for hybridization with a purified ASBV-RNA preparation dot spotted on nitrocellulose paper. Positive self-hybridization indicated a high degree of internal complementarity. Dot spots of whole cell RNA and of leaf sap from ASBV infected plants were shown to hybridize with the labelled probe. This hybridization procedure proved to be 16–64 times more sensitive in diagnosing ASBV when compared with polyacrylamide gel analysis.     

Rapid detection and identification of Vibrio anguillarum by using a specific oligonucleotide probe complementary to 16S rRNA.

Partial 16S rDNA from Vibrio collection type strains and recent isolates of Vibrio-related strains were sequenced and compared with previously published sequences. A 24-base DNA oligonucleotide (VaV3) was designed and used as a specific probe for detection and identification of Vibrio anguillarum. Its specificity was tested against collection type strains and environmental isolates and no cross-reaction was found. The probe detected 8 of the 10 V. anguillarum serovars. It was applied to screen different Vibrio-related strains isolated from marine hatcheries and fish farms. The detection limit in DNA-DNA slot blot hybridization was 150 pg.     

Quantitation of casein messenger ribonucleic acid sequences using a specific complementary DNA hybridization probe.

Two highly purified rat casein mRNA fractions were used as templates to synthesize complementary DNA (cDNA) hybridization probes using RNA-directed DNA polymerase isolated from avian myeloblastosis virus. Both of the probes selectively hybridized to RNA isolated from lactating mammary tissue, but not to poly(adenylic acid)-containing rat liver RNA. An analysis of the kinetics of hybridization of the cDNA derived from the 15S casein mRNA (cDNA12S) with their individual mRNA templates indicated that greater than 90% hybridization occurred over a R0t range of one and one-half logs with R0t 1/2 values of 0.0023 and 0.0032 mol s l.-1, respectively. Compared with the total RNA isolated from lactating mammary tissue, these values represented a 166- and 245-fold purification, respectively, of these individual mRNA fractions. Using the 15S casein mRNA as a template, two probes of different lengths and specific activities were synthesized. The deoxyribonucleotide and mRNA concentrations and the temperature of incubation were optimized to obtain either a high specific activity cDNA probe, 330 nucleotides long, which represented approximately 25% of the mRNA or a lower specific activity preparation containing some complete cDNA copies, 1300 nucleotides in length. The Tm of the longer cDNA15S-15S mRNA hybrid was 88.5 degrees C, while that of the short cDNA15S-RNA hybrid was 82.5 degrees C. Following this initial characterization, the cDNA15S probe was utilized for three separate determinations: (1) Analysis of the sequence divergence between mouse and rat casein mRNAs. It was observed that the rate of hybridization of heterologous rat cDNA15S-mouse casein mRNA was only 20% that of the homologous rat cDNA15S-rat casein mRNA hybridization. The resulting heterologous hybrid displayed approximately 17% mismatching compared with the homologous hybrid. (2) Determination of the gene dosage for casein mRNA in normal and malignant mammary cells. In this study, an analysis of the kinetics of hybridization of the high specific activity cDNA15S probe with an excess of DNA isolated from lactating mammary tissue, carcinogen-induced mammary tumors, or rat liver indicated that casein mRNA was transcribed from the nonlification or deletion was observed during tumor formation or the process of mammary differentiation. (3) Quantitation of casein mRNA sequences during normal mammary gland development. RNA excess hybridizations were performed using RNA extracted from either pregnant, lactating, or regressed rat mammary tissue. The concentration of casein mRNA molecules/alveolar cell was found to increase 12-fold from 5 days of pregnancy until 8 days of lactation and then declined to approximately 2% of the maximal level of 79 000 molecules/cell by 7 days after weaning. A coordinate increase was observed in casein mRNA sequences detected by cDNA hybridization and mRNA activity measured in a cell-free translation assay.     

Specific stabilization of DNA G-quadruplex structures with a chemically modified complementary probe

The DNA G-quadruplex is an important higher-order structure formed from guanine-rich DNA sequences. There are many molecules which can stabilize this structure. However, the selectivity of these ligands to different G-quadruplexes was not satisfactory. Herein, we designed and synthesized a chemically modified G-quadruplex probe, Razo-DNA, for the unique stabilization of the G-quadruplex. Razo-DNA consists of two fragments: The first is an organic molecular moiety which can stabilize G-quadruplex structures, and the second is a DNA molecule that is complementary with a sequence adjacent to the guanine-rich sequence of targeted DNA. Further studies showed that Razo-DNA could precisely stabilize the targeted DNA G-quadruplex structures in vitro.     

Synthesis and characterization of a DNA probe complementary to rat liver 28S ribosomal RNA.

Conditions for the production of a complementary DNA sequence for use in studies of ribosomal RNA are described. $\text{E}$. $\text{coli}$ DNA polymerase I is used to transcribe highly purified 28S ribosomal RNA from rat liver. The reaction is sensitive to the tertiary structure of the rRNA template-primer. The complementary DNA hybridizes to its rRNA template with a $\text{R}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 0.02. The hybrid formed between 28S ribosomal RNA and complementary DNA has a T_m of 73°C. The probe reacts with total rat nuclear RNA with a $\text{R}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 1.0.     

Development of a DNA probe using differential hybridization to detect the fish pathogenVibrio anguillarum

The fish pathogenVibrio anguillarum causes significant economic losses in commercially cultured fish species worldwide. At present, identification ofV. anguillarum requires conventional isolation and culturing techniques. Using differential hybridization, a 310 base pairV. anguillarum-specific DNA fragment was isolated for use as a probe. In specificity studies against 19 different bacterial species, including twoVibrio sp. and fish pathogens, and 223 marine bacterial isolates, the probe hybridized exclusively toV. anguillarum strains. The probe also strongly hybridizes to 7 of 9 serotypes tested, with serotype 09 giving a weak probe reaction and serotype O7 negative. The probe allows rapid and accurate detection of both pathogenic and environmental strains ofV. anguillarum.     

Electrochemical detection of mismatched DNA using a MutS probe

A direct and label-free electrochemical biosensor for the detection of the protein–mismatched DNA interaction was designed using immobilized N-terminal histidine tagged Escherichia coli. MutS on a Ni-NTA coated Au electrode. General electrochemical methods, cyclic voltammetry (CV), electrochemical quartz crystal microbalance (EQCM) and impedance spectroscopy, were used to ascertain the binding affinity of mismatched DNAs to the MutS probe. The direct results of CV and impedance clearly reveal that the interaction of MutS with the CC heteroduplex was much stronger than that with AT homoduplex, which was not differentiated in previous results (GT > CT > CC ≈ AT) of a gel mobility shift assay. The EQCM technique was also able to quantitatively analyze MutS affinity to heteroduplexes.     

Rapid methods of detecting the target molecule in immunohistology using a bioluminescence probe

We demonstrate a novel rapid direct detection method for immunohistochemistry, using a bioluminescent probe. An anti‐CEA antibody‐fused far‐red bioluminescent protein can monitor the accumulation of this type of probe in tumour tissues. The bimodal spectrum (λ_max = 460 and 675 nm) of this bioluminescent probe is extremely stable under different conditions of pH and ion concentration. The sensitivity of our bioluminescent labelling was at the same level of enzymatic labelling, e.g. peroxidase, as an indirect system. Our novel technique is simple and can shorten the pretreatment time of paraffin sections to around 30 min. The utility of our bioluminescent labelling covers all imaging in vitro, in vivo and ex vivo, suggesting that our antibody‐fused bioluminescent probe has the potential to detect tumour antigens with a high sensitivity in routine immune histological examinations. Copyright © 2012 John Wiley & Sons, Ltd.     

Bioassay for specific DNA sequences using a non-radioactive probe

A novel method for detecting specific DNA sequences is described. The method uses a non-radioactive DNA probe, called a probe-vector, that can transform competent Escherichia coli cells at high efficiency only when it has hybridized to a specific DNA target, thus forming a circular, double-stranded, plasmid-like molecule. The probe-vector carries a plasmid origin of replication and a gene that confers antibiotic resistance on transformed E. coli. The output of the assay--colored bacterial colonies on an agar plate--is quantitative and proportional over a wide range of target concentrations. The utility of the probe-vector method for detecting hepatitis B virus (HBV) DNA in human serum is demonstrated. The assay can detect as little as 0.1 pg HBV DNA. The presence of an internal standard monitors DNA recovery and E. coli transformation efficiency for each sample. The assay has the potential to simultaneously measure the DNA of two or more pathogens within the same clinical sample.     

Reiteration of DNA complementary to a cytoplasmic non-ribosomal RNA

Experimentally induced granulomas, in guinea pigs, were fractionated into a 40,000xg pellet and supernatant, which was further fractionated into a 100,000xg pellet and supernatant. The product of in vitro amino acid incorporation by the 40,000xg pellet was tentatively identified as collagen by its high proline/leucine ratio, its digestibility by bacterial collagenase and its solubility in hot trichloroacetic acid. The 100,000xg pellet incorporated leucine much more efficiently than the 40,000xg pellet and the product was insoluble in hot trichloroacetic acid.Labeled RNA from the 40,000xg pellet formed hybrids with granuloma ($\text{C}{}_{\mathrm{o}}\text{t}\text{1}\text{2}\text{= 100–150}$) and liver ($\text{C}{}_{\mathrm{o}}\text{t}\text{1}\text{2}\text{= 8,000}$) $\text{chromatin}$ DNA, indicating that genes coding for this RNA are repeated about 100-fold in granuloma and less than 5 times in liver DNA. Under conditions of poly(A) binding, 50% of this labeled RNA is retained by filters. Digestion with ribonuclease and ribonuclease T₁, decreases binding efficiency by 75%.