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In nucleic acid nanotechnology, strand displacement is a widely used mechanism where one strand from a hybridized duplex is exchanged with an invading strand that binds to a toehold, a single-stranded region on the duplex. It is used to perform logic operations on a molecular level, initiate cascaded reactions, or even for in vivo diagnostics and treatments. While systematic experimental studies have been carried out to probe the kinetics of strand displacement in DNA with different toehold lengths, sequences, and mismatch positions, there has not been a comparable investigation of RNA or RNA-DNA hybrid systems. Here, we experimentally study how toehold length, toehold location (5' or 3' end of the strand), and mismatches influence the strand displacement kinetics. We observe reaction acceleration with increasing toehold length and placement of the toehold at the 5' end of the substrate. We find that mismatches closer to the interface of toehold and duplex slow down the reaction more than remote mismatches. A comparison of RNA and DNA displacement with hybrid displacement (RNA invading DNA or DNA invading RNA) is partly explainable by the thermodynamic stabilities of the respective toehold regions, but also suggests that the rearrangement from B-form to A-form helix in the case of RNA invading DNA might play a role in the kinetics.

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

Synthetic protein switches that sequence-specifically respond to oligonucleotide-based input triggers provide valuable tools for the readout of oligonucleotide-based biomolecular systems and networks. Here, we discuss a highly modular approach to reversibly control the DNA-directed assembly and disassembly of a complex between TEM1-β-lactamase and its inhibitor protein BLIP. By conjugating each protein to a unique handle oligonucleotide, the enzyme-inhibitor pair is noncovalently assembled upon the addition of a complementary ssDNA template strand, resulting in inhibition of enzyme activity. Hybridization of an input-oligonucleotide that is complementary to a target recognition sequence in the ssDNA template strand results in the formation of a rigid dsDNA helix that mechanically disrupts the enzyme-inhibitor complex, hereby restoring enzyme activity. Following this noncovalent approach allowed straightforward tuning of the ssDNA template recognition sequence and target oligonucleotide lengths with only a single set of oligonucleotide-functionalized enzyme and inhibitor domains. Using a fluorescent substrate, as little as 10 pM target oligonucleotide resulted in a distinguishable increase in enzyme activity.

A newly developed coarse-grained model called BioModi is utilized to elucidate the effects of temperature and concentration on DNA hybridization in self-assembly. Large-scale simulations demonstrate that complementary strands of either the tetrablock sequence or randomized sequence with equivalent number of cytosine or guanine nucleotides can form completely hybridized double helices. Even though the end states are the same for the two sequences, there exist multiple kinetic pathways that are populated with a wider range of transient aggregates of different sizes in the system of random sequences compared to that of the tetrablock sequence. The ability of these aggregates to undergo the strand displacement mechanism to form only double helices depends upon the temperature and DNA concentration. On one hand, low temperatures and high concentrations drive the formation and enhance stability of large aggregating species. On the other hand, high temperatures destabilize base-pair interactions and large aggregates. There exists an optimal range of moderate temperatures and low concentrations that allow minimization of large aggregate formation and maximization of fully hybridized dimers. Such investigation on structural dynamics of aggregating species by two closely related sequences during the self-assembly process demonstrates the importance of sequence design in avoiding the formation of metastable species. Finally, from kinetic modeling of self-assembly dynamics, the activation energy for the formation of double helices was found to be in agreement with experimental results. The framework developed in this work can be applied to the future design of DNA nanostructures in both fields of structural DNA nanotechnology and dynamic DNA nanotechnology wherein equilibrium end states and nonequilibrium dynamics are equally important requiring investigation in cooperation.

Potential guanine (G) quadruplex-forming sequences (QFSs) found throughout the genomes and transcriptomes of organisms have emerged as biologically relevant structures. These G-quadruplexes represent novel opportunities for gene regulation at the DNA and RNA levels. Recently, the definition of functional QFSs has been expanding to include a variety of unconventional motifs, including relatively long loop sequences (i.e., >7 nucleotides) separating adjacent G-tracts. We have identified a QFS within the 25S rDNA gene from Saccharomyces cerevisae that features a long loop separating the two 3'-most G-tracts. An oligonucleotide based on this sequence, QFS3, folds into a stable G-quadruplex in vitro. We have studied the interaction between QFS3 and several loop mutants with a small, homologous (G-rich) peptide nucleic acid (PNA) oligomer that is designed to form a DNA/PNA heteroquadruplex. The PNA successfully invades the DNA quadruplex target to form a stable heteroquadruplex, but with surprisingly high PNA:DNA ratios based on surface plasmon resonance and mass spectrometric results. A model for high stoichiometry PNA-DNA heteroquadruplexes is proposed, and the implications for quadruplex targeting by G-rich PNA are discussed.

DNA hybridization, wherein strands of DNA form duplex or larger hybrids through noncovalent, sequence-specific interactions, is one of the most fundamental processes in biology. Developing a better understanding of the kinetic and dynamic properties of DNA hybridization will thus help in the elucidation of molecular mechanisms involved in numerous biochemical processes. Moreover, because DNA hybridization has been widely adapted in biotechnology, its study is invaluable to the development of a range of commercially important processes. In this Account, we examine recent studies of the kinetics and dynamics of DNA hybridization, including (i) intramolecular collision of random coil, single-stranded DNA (ssDNA), (ii) nucleic acid hairpin folding, and (iii) considerations of DNA hybridization from both a global view and a detailed base-by-base view. We also examine the spontaneous single-base-pair flipping in duplex DNA because of its importance to both DNA hybridization and repair. Intramolecular collision of random coil ssDNA, with chemical relaxation times ranging from hundreds of nanoseconds to a few microseconds, is investigated both theoretically and experimentally. The first passage time theory of Szabo, Schulten, and Schulten, which determines the average reaction time of the intrachain collision, was tested. Although it was found to provide an acceptable approximation, a more sophisticated theoretical treatment is desirable. Nucleic acid hairpin folding has been extensively investigated as an important model system of DNA hybridization. The relaxation time of hairpin folding and unfolding strongly depends on the stem length, and it may range from hundreds of microseconds to hundreds of milliseconds. The traditional two-state model has been revised to a multistate model as a result of new experimental observations and theoretical study, and partially folded intermediate states have been introduced to the folding energy landscape. On the other hand, new techniques are needed to provide more accurate and detailed information on the dynamics of DNA hairpin folding in the time domain of sub-milliseconds to tens of milliseconds. From a global view, the hybridization of unstructured ssDNA goes through an entropy-controlled nucleation step, whereas the hybridization of ssDNA with a hairpin structure must overcome an extra, enthalpy-controlled energy barrier to eliminate the hairpin. From a detailed base-by-base view, however, there exist many intermediate states. The average single-base-pair hybridization and dehybridization rates in a duplex DNA formation have been determined to be on the order of a millisecond. Meanwhile, accurate information on the early stages of hybridization, such as the dynamics of nucleation, is still lacking. The investigation of spontaneous flipping of a single base in a mismatched base pair in a duplex DNA, although very important, has only recently been initiated because of the earlier lack of suitable probing tools. In sum, the study of DNA hybridization offers a rich range of research opportunities; recent progress is highlighting areas that are ripe for more detailed investigation.

Background: One of the most fascinating discoveries in biology in recent years is unquestionably the identification of the family of small, noncoding RNAs known as microRNAs (miRNAs). Each miRNA targets multiple mRNA species through recognition of complementary sequences, typically located at multiple sites within the 3 untranslated region. In animals, single-stranded miRNA binds specific messenger RNA (mRNA) by a mechanism that is yet to be fully characterized. The bound mRNA remains untranslated resulting in reduced levels of the corresponding protein; however, if the sequence match between the miRNA and its target is precise, the bound mRNA can be degraded resulting in reduced levels of the corresponding transcript. Eukaryotic genes are also regulated by triplex formation between double helix and a third small RNA or DNA molecule. Thousands of triplex-forming (TF) islands in human genomes are mapped. However, the role of TF miRNAs within the hairpin structures of miRNA and the target mRNA has not been reported. We have explored TF complexes between human miRNAs (hsa-miR) that are complementary to human immunodeficiency virus (HIV)-1 and their antiviral potential as therapeutic agents., Methods: We downloaded mature miRNA sequences from the human miRBase Sequence Database (http://microrna.sanger.ac.uk/sequences/), and computationally analyzed miRNAs that have significant homologies to HIV-1 genome (pNL 4-3 Accession #AF324493). We developed an algorithm to look for triplex-binding motifs (C+CG and T AT) and selected 4 miRNAs with 3 negative controls. TF stability was tested by using fluorophore-labeled duplexes connected by a single hexaethylene glycol moiety, representing HIV-1 proviral motifs, and black-hole quencher-1 labeled oligonucleotides, representing miRNA., Results: Fifty miRNAs were discovered that showed greater than 80% homology to HIV-1, of which 4 hsa-miR that exhibited an ability to form stable triplex with double stranded-HIV-1 sequences were selected. Three negative controls were used. The TF stability of the 4 hsa-miRs and the negative controls were confirmed and measured. Stably transfected Hela-CD4+ cell lines expressing each of the hsa-miR were developed. All 4 miRNAs exhibited a significant inhibition of HIV-1 as measured by HIV-1 p24 enzyme-linked immunosorbent assay (>90%; P>0.001) when compared with the 3 negative controls. By using immunohistochemical staining with triplex binding monoclonal antibodies, significant expression of TF miRNAs was detected in the cell lines, but not in the negative controls (P<0.001)., Conclusions: In this study, we demonstrated for the first time that besides the well-established post-transcriptional silencing based on mRNA degradation, miRNAs may be responsible for long-term latency of HIV-1 by TF, a different mechanism. We provide a possible molecular mechanism by which HIV-1 homologous miRNAs may impart resistance to HIV-1 and suggest a new miRNA-based therapeutic strategy for HIV-1.

RNA molecules will tend to adopt a folded conformation through the pairing of bases on a single strand; the resulting so-called secondary structure is critical to the function of many types of RNA. The secondary structure of a particular substring of functional RNA may depend on its surrounding sequence. Yet, some RNAs such as microRNAs retain their specific structures during biogenesis, which involves extraction of the substructure from a larger structural context, while other functional RNAs may be composed of a fusion of independent substructures. Such observations raise the question of whether particular functional RNA substructures may be selected for invariance of secondary structure to their surrounding nucleotide context. We define the property of self containment to be the tendency for an RNA sequence to robustly adopt the same optimal secondary structure regardless of whether it exists in isolation or is a substring of a longer sequence of arbitrary nucleotide content. We measured degree of self containment using a scoring method we call the self-containment index and found that miRNA stem loops exhibit high self containment, consistent with the requirement for structural invariance imposed by the miRNA biogenesis pathway, while most other structured RNAs do not. Further analysis revealed a trend toward higher self containment among clustered and conserved miRNAs, suggesting that high self containment may be a characteristic of novel miRNAs acquiring new genomic contexts. We found that miRNAs display significantly enhanced self containment compared to other functional RNAs, but we also found a trend toward natural selection for self containment in most functional RNA classes. We suggest that self containment arises out of selection for robustness against perturbations, invariance during biogenesis, and modular composition of structural function. Analysis of self containment will be important for both annotation and design of functional RNAs. A Python implementation and Web interface to calculate the self-containment index are available at http://kim.bio.upenn.edu/software/.

Recent advances in the study of Schistosoma mansoni genome and transcriptome have led to a better description of the S. mansoni gene complement. In this work, we report the design and use of a new S. mansoni 60-mer oligonucleotide microarray platform with approximately 44,000 probes, based on all publicly available cDNA sequence data for S. mansoni and Schistosoma japonicum. The large number of probes combined with the extensive sequence annotation available allowed a comprehensive approach, where most of the S. mansoni transcriptome is represented. Hybridization with adult worm RNA pointed to a set of genes transcriptionally active in this stage of the parasite's life cycle. Interestingly, a large proportion (43%) of genes for which transcription was detected in adults is comprised of "no match" genes, i.e. S. mansoni genes with unknown function and no identifiable orthologs in GenBank. Moreover, detection of bi-directional transcription for 7% of the active "no match" genes in adults leads us to hypothesize a widespread production of antisense RNA in S. mansoni, with possible regulatory roles.

Since the phenotypes of hybrid progenies involving genes with maternal effects are affected by the genotype of female parent, they cannot reflect the genotypes of individuals. This makes it difficult to develop test cross parents (triple or double recessive lines) for linkage localization and, consequently, hinders the progress in localization researches for these types of genes. In this study, we designed a set of hybridization schemes, the key of which was to make the "maternal-effect" genes homozygous at first, and then the non-maternal-effect genes. Using this scheme, we successfully produced a triple recessive line for genes ch (chocolate), nlw (non-lepis wing) and b-t (maternal brown egg of Tsujitan) on the 13th linkage group and a double recessive line for genes nb (narrow breast) and ki-2 (kidney-shaped egg 2) on the 19th linkage group of Bombyx mori.

We are performing experiments that use fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) to monitor the movement of an individual donor-labeled sliding clamp protein molecule along acceptor-labeled DNA. In addition to the FRET signal sought from the sliding clamp-DNA complexes, the detection channel for FRET contains undesirable signal from free sliding clamp and free DNA. When multiple fluorescent species contribute to a correlation signal, it is difficult or impossible to distinguish between contributions from individual species. As a remedy, we introduce "purified FCS", which uses single molecule burst analysis to select a species of interest and extract the correlation signal for further analysis. We show that by expanding the correlation region around a burst, the correlated signal is retained and the functional forms of FCS fitting equations remain valid. We demonstrate the use of purified FCS in experiments with DNA sliding clamps. We also introduce "single-molecule FCS", which obtains diffusion time estimates for each burst using expanded correlation regions. By monitoring the detachment of weakly-bound 30-mer DNA oligomers from a single-stranded DNA plasmid, we show that single-molecule FCS can distinguish between bursts from species that differ by a factor of 5 in diffusion constant.

RNase H belongs to a nucleotidyl-transferase superfamily, which includes transposase, retroviral integrase, Holliday junction resolvase, and RISC nuclease Argonaute. We report the crystal structures of RNase H complexed with an RNA/DNA hybrid and a mechanism for substrate recognition and two-metal-ion-dependent catalysis. RNase H specifically recognizes the A form RNA strand and the B form DNA strand. Structure comparisons lead us to predict the catalytic residues of Argonaute and conclude that two-metal-ion catalysis is a general feature of the superfamily. In nucleases, the two metal ions are asymmetrically coordinated and have distinct roles in activating the nucleophile and stabilizing the transition state. In transposases, they are symmetrically coordinated and exchange roles to alternately activate a water and a 3'-OH for successive strand cleavage and transfer by a ping-pong mechanism.

We previously showed that a specific kind of mRNA (c-fos) was detected in a living cell under a microscope by introducing two fluorescently labeled oligodeoxynucleotides, each labeled with donor or acceptor, into the cytoplasm, making them hybridize to adjacent locations on c-fos mRNA, and taking images of fluorescence resonance energy transfer (FRET) (A. Tsuji, H. Koshimoto, Y. Sato, M. Hirano. Y. Sei-Iida, S. Kondo, and K. Ishibashi, 2000, Biophys. J. 78:3260-3274). On the formed hybrid, the distance between donor and acceptor becomes close and FRET occurs. To observe small numbers of mRNA in living cells using this method, it is required that FRET fluorescence of hybrid must be distinguished from fluorescence of excess amounts of non-hybridizing probes and from cell autofluorescence. To meet these requirements, we developed a time-resolved method using acceptor fluorescence decays. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay with direct excitation. A combination of Bodipy493/503 and Cy5 was selected as donor and acceptor. When the formed hybrid had a configuration where the target RNA has no single-strand part between the two fluorophores, the acceptor fluorescence of hybrid had a sufficiently longer delay to detect fluorescence of hybrid in the presence of excess amounts of non-hybridizing probes. Spatial separation of 10-12 bases between two fluorophores on the hybrid is also required. The decay is also much slower than cell autofluorescence, and smaller numbers of hybrid were detected with less interference of cell autofluorescence in the cytoplasm of living cells under a time-resolved fluorescence microscope with a time-gated function equipped camera. The present method will be useful when observing induced expressions of mRNA in living cells.

RecA protein of E. coli plays a central regulatory role that is induced by damage to DNA and results in the inactivation of LexA repressor. In vitro, RecA protein binds preferentially to single-stranded DNA to form a nucleoprotein filament that can recognize homology in naked duplex DNA and promote extensive strand exchange. Although RecA protein shows little tendency at neutral pH to bind to RNA, we found that it nonetheless catalyzed at 37 degrees C the hybridization of complementary RNA and single-stranded DNA sequences. Hybrids made by RecA protein at 37 degrees C appeared indistinguishable from ones prepared by thermal annealing. RNA-DNA hybridization by RecA protein at neutral pH required, as does RecA-promoted homologous pairing, optimal conditions for the formation of RecA nucleoprotein filaments. The cosedimentation of RNA with those filaments further paralleled observations made on the formation of networks of nucleoprotein filaments with double-stranded DNA, an instrumental intermediate in homologous pairing in vitro. These similarities with the pairing reaction support the view that RecA protein acts specifically in the hybridization reaction.

The nucleoprotein filament formed by the RecA protein of Escherichia coli on single-stranded DNA catalyzes the hybridization of RNA transcripts with single-stranded DNA sequences at 37 degrees C, in vitro. RecA protein rapidly promotes hybridization, even when noncomplementary RNA is in a millionfold nucleotide excess over hybridizing RNA, and in a thousandfold nucleotide excess over hybridizing single-stranded DNA. Heterologous double-stranded DNA and RecA-coated noncomplementary single-stranded DNA are also poor competitors of RNA transcripts produced in vitro. Since large excesses of noncomplementary RNA fail to inhibit sharply the hybridization reaction by RecA protein under mild, non-degradative conditions, the reaction may be useful in the identification and isolation of transcripts produced in vivo.

The distribution of a polymorphism due to an Adenine to Guanine transition in the ApoAI gene has been studied in 136 women and 108 men, through amplification of the promoter region of the gene and allele-specific oligonucleotide hybridization. The allelic frequencies for the A allele were 0.10, 0.14 and 0.27 in women and 0.08, 0.17 and 0.14 in men for the lowest decile, intermediate group and the highest decile of HDL-cholesterol levels, respectively. Statistical analysis showed that the A allele was associated with high HDL-cholesterol levels in women, but not in men.

Two 18-mer oligonucleotides derived from the sequence of hly, the gene coding for listeriolysin O, were shown to be specific for Listeria monocytogenes in the genus Listeria in colony hybridization tests. The oligonucleotides did not hybridize with any of the bacterial species found in food and co-isolated with Listeria on selective media. They were used in colony hybridization tests for enumeration of L. monocytogenes present in food samples after direct plating on selective media plates. In addition, two 24-mer oligonucleotides, each including the sequence of one of the 18-mers, were successfully used for the PCR-based detection of L. monocytogenes bacilli present in food samples after 48-h enrichment period. Using this technique, as little as 10(2) bacteria per ml of enrichment broth can be detected.

Total genomic DNA isolated by concentrating seawater and mercury-resistant bacteria were hybridized with a mer probe to detect the presence of homologous DNA sequences in marine coastal waters of the Bay of Bengal, India. Coastal water extracts induced with mercury hybridized with the mer operon of transposon Tn501. Most of the mercury-resistant bacteria that volatilized mercury also contained homologous DNA sequences to the mer probe.

Epstein-Barr virus is associated with many diseases. Today, the pathologist may study either by immunohistochemistry or in situ hybridization on tissue sections: EBV genome, EBV messenger RNA, EBV latent and replicative proteins. Several technics can be performed on fixed paraffin-embedded tissue to demonstrate the presence of EBV DNA, EBER-1 RNA, LMP-1 protein. Frozen tissues are required for the study of EBNA-2, ZEBRA and replicating proteins expression. The results, obtained during the study of benign and malignant proliferations always or often associated with EBV, such as infectious mononucleosis, Burkitt's lymphomas, AIDS associated lymphomas, lymphoproliferations in immunocompromised patients, Hodgkin's disease, and some epithelial proliferations, are summarized.

Ribosomal DNA (rDNA) polymorphism was compared to enzyme polymorphism and DNA/DNA hybridization data for the intraspecies differentiation of Providencia alcalifaciens. DNA from 27 strains previously classified into two zymotypes A1 and A2 and discriminated by two levels of DNA/DNA hybridization (delta Tm values of 0 to 1 degree C and 6 to 10 degrees C, respectively) were analysed by Southern blotting for rDNA polymorphism. The ribotypes fell into two ribogroups A1 and A2, which correlated with the corresponding zymotypes. This correlation argues for the proposed creation of two species within P. alcalifaciens.

Subtractive differential hybridization was used to identify genes in Bacillus subtilis that are induced by nutrient limitation. Several transcription units were identified. They exhibited increased transcription when cells were deprived of certain nutrients, such as glucose, ammonium, or phosphate, or when cells were treated with decoyinine. The genes have been designated dci (for decoyinine-inducible) and gsi (for glucose-starvation-inducible). Using lacZ transcriptional fusions, the dependence of dci and gsi expression on gene products of the sensor and activator classes of bacterial two-component regulatory systems was examined. Transcription of dciA was impaired by a mutation in spoOA, while expression of gsiA was dependent on the early competence genes comP and comA. The implications of these findings are discussed, and a provisional scheme for information flow during the transition phase from growth to sporulation is proposed.

We used the Bacillus subtilis dnaB37 mutant, which is defective in initiation, to synchronize DNA replication in order to identify the first fragments to be replicated following initiation and to study the control of this process under various conditions. We show by DNA/DNA hybridization analysis that, after returning the mutant from 45 degrees C to the permissive temperature (30 degrees C), the origin region relative to other sequences is over-replicated (approximately 2-fold) during the first round. This was confirmed by autoradiographic analysis. The over-replicated region is however limited to about 190 kb on the left and right arms. Replication apparently resumes from these positions during the following round of replication. We propose that, in B. subtilis, in addition to the first level of control at the origin, there is a second level or post-initiation control downstream of the origin which limits DNA replication resulting from premature initiation. We believe that these two levels of control are tightly coupled under conditions of balanced growth. Using the same system, we have now shown that DNA replication is subject to "stringent control", an important regulatory network in bacteria. These studies demonstrate that the inhibition of replication induced during the "stringent response" does not occur at the primary origin. In fact, by DNA/DNA hybridization, replication forks were found to be blocked at similar positions to the post-initiation control sites described above. Moreover, replication appears to resume from regions close to the stalled replisomes upon removal of the stringent response.(ABSTRACT TRUNCATED AT 250 WORDS)

Vector plasmids were constructed by ligating chloramphenicol and erythromycin resistance genes to TaqI-digested DNA of a cryptic plasmid from Lactobacillus plantarum. The minimal region of Lactobacillus plasmid DNA that was required for DNA replication was defined and a single-stranded DNA intermediate replication system was observed. Homologies with other origins of replication of plasmids from Gram-positive bacteria, replicating via rolling circle mechanism, were found. It was shown that the constructed vectors, named pPSC20 and pPSC22, were transformable into L. plantarum, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus helveticus, Lactococcus lactis subsp. lactis, Bacillus subtilis, and Escherichia coli. Using plasmid pPSC22, the alpha-amylase gene of Bacillus stearothermophilus was cloned and expressed in several Lactobacillus species.

The genomes of 18 independent Shigella isolates (9 Shigella sonnei, 5 Shigella dysenteriae and 4 Shigella flexneri) as well as of 4 epidemic S. flexneri strains were analysed by pulsed field gel electrophoresis (PFGE) and by the distribution of insertion sequences (IS1, IS2 and IS911). Despite the close relatedness observed among the 9 independent S. sonnei, all of them could be differentiated from each other. The 4 independent S. flexneri isolates showed clearly distinguishable DNA profiles. Nearly complete genetic identity was detected within the 4 epidemic S. flexneri when analysed by PFGE or for IS1 and IS2 patterns. However, IS911 was found to be too mobile in these epidemic S. flexneri to be used as a typing probe. The 5 S. dysenteriae isolates could also be distinguished by the techniques used. The diversity found within this species is striking: of the 5 investigated isolates, 3 completely different DNA profiles were revealed. In conclusion, both PFGE and IS probing demonstrated their potential usefulness in molecular epidemiology and in typing of Shigella strains. The degree of differentiation given by these two methods was generally comparable, although IS probes showed better discrimination of the isolates.

Seventeen suspension of Legionella pneumophila and ten of Legionella bozemanii in saline or bronchoalveolar lavage (BAL) fluid were tested using the Gen Probe technique. The detection threshold was found to be 10(3)-10(4) CFU/ml. Specificity and sensitivity were evaluated by using the probe on 8 suspensions of bacteria other than Legionella and by performing a comparative study of the probe test, direct immunofluorescence and culture with 103 specimens (BAL fluid in most instances) from 92 patients with possible legionellosis. Sensitivity was found to be acceptable (3 of the 4 culture-positive specimens were positive by the probe test) and specificity was 100% despite the fact that most (80/99) BAL specimens were not sterile and regardless of the cutoff level used to define positivity. The advantages of the DNA probe test, including rapidity, simplicity and objectivity, should be weighed against its disadvantage, i.e., only acceptable sensitivity and use of radioactivity.

The increased level of IL-1 mRNA was discovered in neutrophils of patients suffering from pollinosis for a long time and responding poorly to specific immunotherapy. Moreover, neutrophils from patients with exacerbation of pollinosis responded to stimuli less than cells from healthy donors. In remission, the activated neutrophils from pollinosis patients and healthy donors had the same levels of IL-1b mRNA. Tumor necrosis factor alpha expression was unchanged in neutrophils of pollinosis patients. Changes in the status of cells from patients were detected with a simple procedure of dot hybridization. Peripheral blood cells were used as available material. This allows one to hope that the approach can be used for analyzing the patients' immune status in choosing the treatment course as well as for controlling the treatment effect.

The distribution of mRNA expression for three types of voltage gated neuronal sodium-channels was studied in the rat brain at different developmental stages (embryonal day E18, postnatal day P5 and adult). With the in-situ hybridization technique, using synthetic DNA-oligomer probes, pronounced regional and temporal variations in the expression levels of the different channel subtypes could be detected. In comparison with types I and III, sodium channel II mRNA was the most abundant subtype at all developmental stages. Maximal expression of sodium channel II mRNA was seen at P5 in virtually all parts of the grey matter, except for the cerebellum. In adult rat brain in contrast, sodium channel II mRNA levels were maximal in the granular layer of the cerebellum, whereas in all other regions expression had decreased to roughly 50% of postnatal levels. Na channel I expression was virtually absent at E18 and showed highest levels at P5, with maxima in the caudate nucleus and hippocampus. In the adult brain, expression of Na-channel I was nearly absent in the neocortex, but well detectable in the cerebellum and, at lower levels in the striatum and thalamus. Sodium channel III was mainly expressed at the embryonal stage and showed a decrease to very low levels with little regional preferences in the adult.

We studied distribution of mRNA for nuclear protooncogene c-myc and nuclear protein P-53 in mature oocytes and embryos of Xenopus laevis from the stage of fertilization up to the stage of hatching by in situ hybridization with histological sections. mRNA for c-myc was present in all cells of the embryo at all studied developmental stages. Between the stage of fertilization and up to the late blastula, mRNA concentration for c-myc decreased progressively in all embryonic cells. During gastrulation a local increase in the concentration of this messenger was found in dorsal mesoderm and ectoderm. At the stage of neurula increased concentration of mRNA for c-myc was observed in all cells of the embryo but the hybridization signal increased particularly distinctly in cells of the neural tube. In studies of P-53 mRNA distribution hybridization signal was detected only in brain cells after stage 20 of development (after closure of the neural folds) and up to the stage of hatching.

In situ hybridization histochemistry technology was developed for future application to neuro-otologic research. This method allowed the detection of cellular mRNA in tissue sections from the temporal bone or brainstem after cRNA/mRNA hybridization. To produce specific cRNA, single-stranded 35S-labeled cRNA (complimentary to target mRNA) is transcribed from commercially available plasmid vectors. These vectors contain promotor sequences for specific synthesis of RNA, and polylinker regions that will accept cloned DNA inserts for virtually any target nucleic acid sequence of interest. The protocol used in this research was optimized for studies that included concomitant immunohistochemical evaluation. The combination of in situ hybridization and immunohistochemistry provides the only method to correlate molecular information (gene expression) with biochemical or molecular markers, such as peptides or proteins (mRNA translation products) on individual cells in the temporal bone or brainstem. Using these techniques, we examined the distribution of the neuropeptide calcitonin gene-related peptide in rat temporal bone and brainstem sections using calcitonin gene-related peptide (CGRP) antisera and CGRP cRNA probes. We used in situ hybridization histochemistry with a cRNA probe complementary to the 3'-end noncoding sequence of the alpha CGRP mRNA and immunohistochemistry with a polyclonal antibody to the (TYR)CGRP23-37 to study the distribution of CGRP mRNA and CGRP-like immunoreactivity in the central and peripheral facial nerve. Numerous motoneuron cell bodies in the facial nucleus and accessory seventh nucleus and cell bodies in the gustatory geniculate ganglion were found to contain CGRP mRNA and the CGRP peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

A low productive laboratory strain of S. antibioticus and a strain with an increased productivity of oleandomycin derived from it were studied comparatively with using restriction analysis and blotting hybridization. Amplification, site specific integration and segregation of the DNA sequence 32.0 kb in size were detected in the strains. The chromosomes of the laboratory strain contained one copy of the amplifying sequence AUD. After uniting of the end sequences AUD appeared to be capable of segregating from the chromosomes and its one copy per five genomes was present in the form of an extrachromosomal genetic element eSA1. The genome of the strain with increased productivity of oleandomycin contained in its chromosomes sequence ADS-Sa1 amplified to 150 copies and the eSA1 extrachromosomal genetic element in the form of mono-, di- and trimeric structures in the quantity of approximately one copy per genome. The BamHIB fragment of the eSA1 DNA 4 kb in size was identified. The fragment was able to participate in segregation or integration of eSA1 from or into the chromosomes since its subfragments were flanking AUD and ADS-SA1 in the chromosomes. The BamHIB fragment was hybridizing with a number of fragments of the chromosomal DNA of S. antibioticus, S. erythraeus. S. lividans and other strains of streptomycetes. It probably contained an IS-like element or a dispersed genetic element of another class. The DNA sequence of the eSA1 genetic element contained regions homologous to the sequence of the Erm E gene in S. erythraeus NRRL 2338.

An unusual variant of chromosome 6 was shown to be a dic(6) by use of the chromosome specific alphoid probe, p308. The use of biotinylated repeat probes provides a rapid procedure for the resolution of some cytogenetic abnormalities.

Mitochondrial RNA biosynthesis was studied at different time after hydrocortisone administration. Hydrocortisone induced liver mitochondrial RNA biosynthesis in vivo and in isolated organellae 15 min., 1.5 and 4 h after hormone injection. As shown by means of molecular hybridization of RNA-DNA, an increase in liver mitochondrial RNA biosynthesis induced by hydrocortisone administration was due to an increase in the expression of all mitochondrial genes simultaneously rather than to selective enhancement of individual gene transcription. Hydrocortisone changed the biosynthesis and transport of cytoplasmic RNA in the liver of intact rats. The incorporation of 3H-UTP into RNA by isolated liver mitochondria of control and hydrocortisone-treated rats was inhibited by ethidium bromide. RNA biosynthesis in isolated rat liver mitochondria was unchanged in 15-300 min. of 0.9% NaCl administration.

The effects of 1,25(OH)2D3 and dexamethasone on cellular proliferation and gene expression of the HTLV-I-infected T-cell line, KH-2, established from a patient with adult T-cell leukemia, endemic in the south-west Japanese islands and the Caribbean, were examined. KH-2 cells are integrated by HTLV-I proviral DNA and expressed mRNA for c-myc, IL-2 receptor alpha-chain (IL-2R alpha), and T-cell receptor beta-chain (TCR beta) while it did not express IL-2 mRNA. 1,25(OH)2D3 and dexamethasone did not suppress the mRNA levels of HTLV-I, IL-2R alpha or TCR beta but reduced the c-myc mRNA level. The reduction of c-myc mRNA level was marked in 1,25(OH)2D3-treated cells but relatively weak in dexamethasone-treated cells. This inhibitory effect of the steroid hormones correlated with the inhibition of KH-2 cell proliferation.

The Chemiprobe Kit provides a complete system for nonradioactive labeling of DNA probes and their detection in hybridization studies. The system is highly sensitive, permitting the detection of 0.2-0.4 pg DNA which allows detection of a single gene sequence in 0.5-1 microgram of bacterial DNA or in 3-5 micrograms of mammalian DNA. In this paper the authors show that the rRNA genes of M. capricolum can be detected by using only 50 ng/ml of sulfonated probe cloned from another mycoplasma, M. pneumoniae. The Chemiprobe system has been successfully used in the detection of the single copy human gene for glucocerobrosidase from total embryonic DNA by hybridization to a specific sulfonated cDNA. 5 x 10(4) M. pneumoniae cells can be detected either free or mixed with sputum using a standard dot blot technique: mycoplasma cells were lysed by a mucolytic agent, denaturated by NaOH, immobilized on a nylon membrane filter, and then hybridized with pPN4, a plasmid DNA probe specific for M. pneumoniae. The resulting hybrids were then detected by the standard Chemiprobe procedure. A new kit based on the Chemiprobe system has been designed especially for the detection of mycoplasmas in tissue culture. This kit has been tested on 70 random samples collected from tissue culture fluids from 11 different sources. Of these, 42 were found to be contaminated by the Chemiprobe procedure, whereas 41 were found to be contaminated by classical microbiological methods. No false negatives were found.