A Simple RNA Probe System for Analysis of Listeria monocytogenes Polymerase Chain Reaction Products.
[This corrects the article on p. 2796 in vol. 59.].
Journal Article↗
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Probing RNA tertiary structure: interhelical crosslinking of the hammerhead ribozyme.
Distinct structural models for the hammerhead ribozyme derived from single-crystal X-ray diffraction and fluorescence resonance energy transfer (FRET) measurements have been compared. Both models predict the same overall geometry, a wishbone shape with helices II and III nearly colinear and helix I positioned close to helix II. However, the relative orientations of helices I and II are different. To establish whether one of the models represents a kinetically active structure, a new crosslinking procedure was developed in which helices I and II of hammerhead ribozymes were disulfide-crosslinked via the 2' positions of specific sugar residues. Crosslinking residues on helices I and II that are close according to the X-ray structure did not appreciably reduce the catalytic efficiency. In contrast, crosslinking residues closely situated according to the FRET model dramatically reduced the cleavage rate by at least three orders of magnitude. These correlations between catalytic efficiencies and spatial proximities are consistent with the X-ray structure.
Base Sequence↗
PCR-based preparation of 23S rRNA-targeted group-specific polynucleotide probes.
DNA coding for a variable region within domain III of bacterial 23S rRNA was used as the target for group-specific polynucleotide hybridization probes. The corresponding rDNA was amplified in vitro by the PCR technique in combination with a pair of primers specific for flanking conserved target sites. The amplified fragments were cloned or used directly as probes. RNA probes were generated by in vitro transcription of cloned or amplified rDNA. The probes were labeled by incorporating modified nucleotides during in vitro DNA amplification or in vitro transcription or by random priming. The use of in vitro transcribed single-stranded RNA probes instead of double-stranded DNA probes provided stronger hybridization signals. Group-specific probes were prepared from genomic DNAs or directly from cells of Acinetobacter calcoaceticus, Alcaligenes faecalis, Aeromonas hydrophila, Nannocystis exedens, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas stutzeri.
Bacteria↗
Infection of human fetal dorsal root ganglion glial cells with human immunodeficiency virus type 1 involves an entry mechanism independent of the CD4 T4A epitope.
Human immunodeficiency virus type 1 (HIV-1) has been implicated in the generation of acquired immunodeficiency syndrome-associated neurological dysfunction, and it is believed that the presence of CD4 in the nervous system may be involved in the susceptibility of selected neural cell populations to HIV-1 infection. We previously demonstrated (B. Wigdahl, R. A. Guyton, and P. S. Sarin, Virology 159:440-445, 1987) that glial cells derived from human fetal dorsal root ganglion (DRG) are susceptible to HIV-1 infection and subsequently express at least a fraction of the virus genome. In contrast to HIV-1 infection of CD4+ lymphocytes, which can be blocked by treatment with monoclonal antibodies directed against the HIV-1-binding region of CD4 (T4A epitope), treatment of human fetal DRG glial cells with similar antibodies resulted in only a slight reduction in HIV-1-specific gag antigen expression. In addition, preincubation of the HIV-1 inoculum prior to infection with HIV-1-neutralizing antiserum did not reduce HIV-1 gag antigen expression in these cells. Furthermore, we were unable to detect the synthesis or accumulation of the CD4 molecule in neural cell populations derived from DRG. However, a protected CD4-specific RNA fragment was detected in RNA isolated from human fetal DRG and spinal cord tissue by an RNase protection assay with a CD4-specific antisense RNA probe. RNA blot hybridization analysis of total cellular RNA isolated from human fetal DRG and spinal cord demonstrated specific hybridization to an RNA species that comigrated with the mature 3.0-kilobase CD4 mRNA as well as two unique CD4 RNA species with relative molecular sizes of approximately 5.3 and 6.7 kilobases. Furthermore, all three CD4-related RNA species were polyadenylated when isolated from human fetal spinal cord tissue. These data suggest that HIV-1 infection of human fetal DRG glial cells may proceed via a mechanism of viral entry independent of the T4A epitope of CD4.
Antibodies, Monoclonal↗
Design, preparation and testing of suitable probe-receptors for RNA biosensing.
Absolute measurements of a given RNA in a cheap, easy, rapid and reproducible manner using biosensors technology could overcome many of the operative and analytical limits of conventional molecular biology methods. To this end, an integrated approach for the design, synthesis, and connection of RNA probes to the transducing surface of a microgravimetric biosensor has been developed. Suitable probes to be used as the bioreceptors in RNA biosensor were successfully designed by using a purposely developed computational method whose selection criteria are based on the accessibility of target region to probe, on pairing stability of probe-target duplex and on the uniqueness of selected targets over all known expressed sequences from a genome data base. Automated chemical synthesis of selected probes was performed and the oligonucleotides produced were covalently conjugated to the sensing surface of a quartz microbalance. The microgravimetric sensor was tested in a flow chamber by measuring the variation of resonance frequency due to the binding of synthetic target substrates. Specific dose dependent binding was observed. Furthermore, the binding of a transcribed full-length mRNA substrate was successfully monitored under similar conditions.
Base Sequence↗
Detection of parvovirus DNA in human serum using biotinylated RNA hybridisation probes.
Methods were established for the detection of parvovirus DNA in human serum using single-stranded RNA probes. The sensitivity of detection of virus using 32P-radiolabelled RNA versus non-radiolabelled biotinylated RNA probes using a streptavidin-polyalkaline phosphatase detection system was compared. Virus was detected using 32P-labelled and biotinylated RNA probes at serum dilutions of 10(-3), equivalent to approx. 3 pg of viral DNA. Using biotinylated RNA probes and a dot-blot system, diagnosis of numerous serum samples could be performed within 8 h of receipt of samples, using an RNA probe which was synthesised and stored at -20 degrees C for up to 12 months without loss of sensitivity. Our work demonstrates the potential of biotinylated RNA probes in the routine analysis of viral sequences in serum.
DNA, Viral↗
Preparation of probe-modified RNA with 5-mercapto-UTP for analysis of protein-RNA interactions.
We report a modified synthesis for 5-mercapto-UTP (5-SH-UTP) and its use for analysis of protein-RNA interactions utilizing Escherichia coli and T7 RNA polymerases and yeast RNA polymerases I and III. 5-SH-UTP did not affect transcriptional pausing, Rho-independent termination or recognition of the E. coli transcription complex by NusA. RNA containing 5-SH-UMP did not crosslink to polymerase when irradiation was 302 or 337 nm. Transcription complexes containing RNA substituted with 5-SH-UMP were post-transcriptionally modified to attach a photocross-linking group to thiol-tagged nucleotides in the RNA on the surface of the polymerase of free in solution. The pKa for 5-SH-UTP was determined to be 5.6, so modification of the thiol groups in the RNA with p-azidophenacyl bromide could be carried out at pH 7. Addition of the transcription termination factor Rho, a RNA binding protein, to E. coli transcription complexes resulted in RNA crosslinking to Rho and to the beta and beta' subunits of polymerase. Using 5-SH-UTP, one can distinguish RNA binding domains on the surface of RNA polymerases or other RNA binding proteins from those buried within the protein.
Base Sequence↗
[Hybridase cleavage of RNA. I. Oligonucleotide probes for regiospecific RNA cleavage].
New oligonucleotide probes for regiospecific cleavage of RNA molecules by hybridase (RNase H) are suggested. RNase H from E. coli is shown to site-specifically split eight phosphodiester bonds in RNA in the heteroduplex, formed by 5S rRNA and d(ACCACCGCGCT). The partial substitution of deoxycytidines in position 5, 6, 8, 10 of the probe by 2'-O-methylcytidines leads to unique (regiospecific) RNA cleavage between U25 and C26.
Base Sequence↗
In situ hybridization: identification of rare mRNAs in human tissues.
In situ hybridization is used for detection of RNA expression when conservation of tissue architecture is important. Most in situ hybridization protocols are written for tissues from animals (i.e., rat) which can be harvested and preserved rapidly. In contrast, human tissue is more difficult to obtain, hence in situ hybridization experiments must frequently be performed with less than optimal tissue preservation. This procedure details hybridization of a radiolabeled single-stranded RNA probe (riboprobe) to complementary sequences of cellular RNA in human tissue sections. This method enables detection of rare mRNA species in specific cell types of human tissue, offering distinct advantages over other in situ methods due to increased sensitivity. In particular, we have found that UV cross-linking and ribonuclease treatment protocols need to be altered for human tissues to ensure successful results, making this protocol unique to those previously described. In situ hybridization experiments can be performed using either DNA or RNA probes. RNA probes are advantageous since they form stable hybrids, are single-stranded, have little or no reannealing during hybridization, and can be synthesized to high specific activity. RNA probes can be readily created utilizing SP6, T3, or T7 promoters in both sense and antisense orientations to provide non-specific (control) and specific probes. Disadvantages of RNA riboprobes include a tendency for RNA to stick non-selectively more than DNA, and degradation by RNase (hence strict adherence to RNase-free precautions is mandatory during most of the protocol). The following protocol includes: (1) preparation of human tissues (tissue fixation and sectioning are highlighted as critical for probe penetration, preservation of tissue architecture, retention of tissue RNA, and overall success); (2) generation of radiolabeled riboprobes (total incorporation of radionucleotide is important to increase sensitivity; 35S was chosen as a compromise between excellent sensitivity, cellular resolution, and required exposure times (compared with 32P or 3H); non-isotopic methods have not been tested in a side-by-side comparison with 35S in human tissues by us, but theoretically might offer faster exposure times while maintaining high resolution); (3) hybridization conditions (stringency, temperature, washes, tissue dehydration); and (4) sample visualization (application of photographic emulsion, developing, fixing, staining, and counterstaining of individual slides).
Humans↗
RNA structure analysis using methidiumpropyl-EDTA.Fe(II): a base-pair-specific RNA structure probe.
Methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] in the presence of dithiothreitol, is shown to cleave phenylalanine-accepting tRNA (tRNAPhe) in a structure-specific fashion. Molar ratios of MPE.Fe(II) to tRNAPhe of less than 1 preferentially cleave phosphodiester bonds known to occur in double-stranded regions of the tRNAPhe molecule. Microdensitometric analysis of autoradiograms of MPE.Fe(II) cleavage products following gel electrophoresis reveals a correspondence between preferred sites of MPE.Fe(II) cleavage and sites in tRNAPhe most sensitive to cobra venom ribonuclease, a double-strand-specific endoribonuclease. Conversely, sites of cleavage by the single-strand-specific S1 nuclease correspond to those nucleotides that are least susceptible to MPE.Fe(II) hydrolysis. Sensitive helical regions in tRNAPhe include the dihydrouracil and the "T psi C" stems, which cannot be detected by cobra venom ribonuclease because of steric constraints. Phosphodiester bonds within the T psi C and dihydrouracil loop regions, which are not detected by S1 nuclease under rigorously controlled digestion conditions, are revealed by inference from their relative insensitivity to MPE.Fe(II). These results demonstrate the utility of MPE.Fe(II) as a general small molecular weight probe of RNA structure, having a greater accessibility to base-paired regions than do the more bulky enzymic probes.
Base Sequence↗
Chemical and computer probing of RNA structure.
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Base Sequence↗
Identification and localization of insulin-like growth factor-binding protein (IGFBP) messenger RNAs in human hair follicle dermal papilla.
The role of the insulin-like growth factors (IGFs) in hair follicle biology has recently been recognized, although their actions, sites of production, and modulation by the insulin-like growth factor-binding proteins (IGFBPs) have not to date been defined. IGF-I is essential for normal hair growth and development, and may be important in regulation of the hair growth cycle. In many culture systems, IGF-I actions are modulated by the IGFBPs. Thus, if IGFBPs are produced in the human hair follicle, they may play a role in targeting IGF-I to its receptor or may modulate IGF-I action by interaction with matrix proteins. We have used in situ hybridization to localize messenger RNA for the six IGFBPs in anagen hair follicles. Anti-sense and sense RNA probes for the IGFBPs (IGFBP-1 to -6) were produced, and 5-micrometer sections of adult facial skin were probed. Messenger RNA for IGFBP-3, -4, and -5 were identified, with predominantly IGFBP-3 and -5 mRNA found in the dermal papilla, and to a lesser extent IGFBP-4 mRNA. IGFBP-4 mRNA was also found at the dermal papilla/epithelial matrix border. Messenger RNAs for both IGFBP-4 and -5 were also demonstrated in the dermal sheath surrounding the hair follicle. Messenger RNAs for IGFBP-1, -2, and -6 were not identified. These studies demonstrate specific localization of IGFBP mRNAs in hair follicles, suggesting that they each play specific roles in the local modulation of IGF action during the hair growth cycle.
Adult↗
RNA-protein interactions at the 3' end of the hepatitis A virus RNA.
The regulative cis-acting terminal RNA structures and the proteins involved in the amplification of the hepatitis A virus (HAV) genome are unknown. By UV cross-linking/label transfer experiments, we have analyzed sequences of the 3'-nontranslated region (3'NTR) and preceding domains of the viral genome for their ability to interact with host proteins. A series of cDNA constructs were used to create genomic- and antigenomic-sense transcripts. The results show that the 3'-NTR-poly(A) interacted with host cell proteins with molecular masses of 38, 45, 57, 84, and 110 kDa only weakly, compared with RNA structures also consisting of 3D-coding regions. Protein p38 was most efficiently labeled after interaction with secondary-structure elements located at the 3' end of the HAV RNA, p38 also interacted with a 5'-terminal RNA probe. Optimal RNA binding was found to be dependent on the salt concentration. The specificity of the RNA-protein interaction was proven by competition assays. These data might indicate that a higher-order structure formed at the junction of the 3Dpol-coding sequence and the 3'-NTR of the HAV genome (putative RNA pseudoknot) significantly improves binding of host proteins and thus suggests that this structure might be essential for the formation of the replication complex initiating minus-strand RNA synthesis.
Animals↗
Labeling fluorescence in situ hybridization probes for RNA targets.
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Base Sequence↗
Hepatocyte growth factor mRNA in human liver cirrhosis as evidenced by in situ hybridization.
BACKGROUND: Hepatocyte growth factor (HGF) is a strong mitogen of hepatocytes, and HGF-producing cells have been reported to be Ito cells or endothelial cells in the sinusoid of the liver. No reports have been published about the localization of HGF mRNA in human liver cirrhosis. METHODS: In situ hybridization (ISH) for HGF mRNA was performed in 5 normal liver and 16 human liver cirrhosis specimens, using 1 RNA probe and 3 oligonucleotide probes labeled with 35S. RESULTS: A positive signal was obtained in 15 of these cases. In five normal liver specimens, signals of HGF mRNA were not obtainable. In 13 of the 15 cases of liver cirrhosis, HGF mRNA was present in the periphery of the regenerative nodules. This peripheral pattern was seen in regenerative nodules with irregular nodule to septal interfaces. Combined immunohistochemistry and ISH showed that vimentin and CD 68-positive cells consistent with macrophages expressed HGF mRNA in such cases. In three specimens with diffuse signal for HGF mRNA in the hepatic nodules, signals localized to the sinusoidal spaces. HGF mRNA-positive cells were spindled and polygonal in shape, suggesting endothelial, Kupffer, and/or Ito cells of origin. In the diffuse pattern the peripheral margins of the regenerative nodules appeared well-defined. In one case regenerative nodules with both diffuse and peripheral signal patterns were present in the same section. There was no relationship among HGF mRNA, etiology, and macroscopic appearance of liver cirrhosis. CONCLUSIONS: HGF gene transcription in human liver cirrhosis nodules may be heterogeneous, probably related to the degree of activity of the regenerative nodules. HGF appears to be produced by the mesenchymal cells, including Ito cells, macrophages (Kupffer cells), and endothelial cells in human liver cirrhosis.
Antigens, CD↗
Detection of porcine parvovirus using nonradioactive nucleic acid hybridization.
Nonradioactive slot blot hybridization assays were established for the detection of porcine parvovirus (PPV), using either a digoxigenin-labeled DNA probe or a biotinylated RNA probe. All probes were prepared from a 3.3-kb Pst1-EcoR1 DNA fragment of the NADL8 isolate of PPV. The sensitivity and specificity of the probes in a slot blot system were evaluated in comparison with a 32P-radiolabeled RNA probe. Using an anti-digoxigenin alkaline phosphatase detection system, at least 1 ng of viral replicative form (RF) DNA, or the equivalent of 100 plaque forming units (PFU) of infectious virus, could be detected by the digoxigenin-labeled DNA probe. When the biotinylated RNA probe and a strepavidin-alkaline phosphatase detection system were employed, 0.1 ng of RF DNA, or the equivalent of 10 PFU of infectious virus, were detected, comparable to the sensitivity of the 32P-radiolabeled RNA probe. Hybridization was not observed with control DNA samples extracted from swine testicle cells, porcine kidney (PK-15) cells, uninfected mixed swine fetal tissue, or from an unrelated DNA virus (pseudorabies virus) infected PK-15 cells. Different isolates of PPV, namely NADL8, NADL2, KBSH, and Kresse, reacted on an equimolar basis in sensitivity and specificity to the biotinlyated probe. Extraction of DNA directly on the filter membrane (direct filter hybridization) was employed in an attempt to reduce processing time by eliminating DNA extraction steps. Direct filter hybridization was indeed less time consuming; it was also comparable in sensitivity and specificity to those methods employing purified DNA.
Animals↗
Differentiation of Schistosoma haematobium from related species using cloned ribosomal RNA gene probes.
The ribosomal RNA (rRNA) gene units of Schistosoma mansoni (lateral spined eggs) and six species of schistosomes with terminal spined eggs (S. haematobium, S. curassoni, S. bovis, S. intercalatum, S. margrebowiei and S. mattheei) have been studied. The schistosome rRNA gene unit consists of a regular interspersion of the two genes encoding the large and small rRNA units with two spacers. The large spacer is not transcribed while the small spacer is part of the transcription unit. Variation in the rRNA gene unit of the species studied is demonstrated and takes three forms: First, variation in DNA sequence leads to both reduced homology in the spacer regions between species and loss or gain of restriction sites. Second, variation in the length of the transcribed spacer is demonstrated and DNA insertions of 0.2 kilobases (kb) and 0.1 kb are observed in S. mattheei and S. margrebowiei, respectively. Third, the length of the non-transcribed spacer region varies between species. S. haematobium has a 0.5 kb deletion in this region, while that of S. margrebowiei contains varying numbers of a 0.4 kb DNA insert. These interspecific variations have been shown to be conserved within species. Analysis of the rRNA genes by DNA hybridisation techniques therefore serves as a means of species identification, whereby it is possible to differentiate S. haematobium from other schistosome species with terminal spined eggs. Similarly, S. margrebowiei and S. mattheei may be clearly distinguished, although no major variation has been detected between S. curassoni, S. bovis and S. intercalatum. All these species differ from S. mansoni by the absence of certain restriction sites in the non-transcribed spacer.
Animals↗
Combination of in situ hybridization and immunocytochemistry to detect messenger RNAs in identified CNS neurons and glia in tissue culture.
We have developed a technique in which immunofluorescence is combined with in situ hybridization using cDNA and RNA probes to assess the expression and distribution of messenger RNAs (mRNA) by neurons and neuroglia in tissue cultures of the rat dentate gyrus. The probes used in this study include a cDNA probe for ribosomal RNA (rRNA) and an RNA probe (cRNA) for glial fibrillary acidic protein (GEAP), an intermediate filament protein subunit expressed by astrocytes in the central nervous system. Both ubiquitous (tubulin) and cell type-specific (MAP-2 and GEAP) antibodies were used to identify neurons and neuroglia in culture. Using this procedure, the mRNA for rRNA was found in the cell bodies and large processes of MAP-2-positive neurons and throughout the cytoplasm of GEAP-positive flat astrocytes. In process-bearing astrocytes, GEAP mRNA is concentrated in the cell body, although some hybridization also occurred in astrocyte cell processes. With this combined in situ hybridization-immunofluorescence technique, the expression and distribution of an mRNA can be examined in different immunocytochemically identified cell types under identical culture and hybridization conditions. It is also possible to determine if there is a differential subcellular distribution of an mRNA in a single cell and if the distribution of the mRNA reflects the distribution of the protein itself. Finally, this technique can be utilized to verify the specificity of probes for cell type-specific mRNAs and to determine appropriate hybridization conditions to produce a specific signal.
Animals↗