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DNA sequence analysis of a 6-kb pathogenicity island, from113 enterotoxigenic and 191 nontoxigenic Bacterioides fragalis, shows that the specific segment and its flanking regions originate from different mobile genetic elements. This indicates that the enterotoxigenic bacteria may have evolved by horizontal transfer of these two genetic elements.

Results show that affinity purified proteins serine/threonine kinase and serine/threonine phosphoprotein phosphatase are 39 and 27 kDa proteins respectively with the former exhibiting autophosphorylation and the latter dependent on manganese for activity.

Adenosine triphosphate (ATP) has been found to manifest complex saturation kinetics when allowed to interact with single-stranded DNA, as revealed by a coding sequence conducted on Escherichia coli strains. Results further revealed that the nucleoside triphosphate phosphohydrolase I (NPH I) may take the form of conformational states, depending on the level of ATP observed. It was also shown that the use of ultraviolet radiation may trigger a photo-cross link between RNA and DNA, reflecting the presence of a common binding site for both amino acids' oligonucleotides.

An internal standard RNA with a 7-nucleotide deletion for competitive reverse transcription-polymerase chain reaction (RT-PCR) is used in quantifying hepatitis A virus (HAV) in experimentally contaminated mussels. The standard RNA is deposited directly into the sample and is used as an extraction control and as quantification tool. The RT-PCR assay offers a reliable method for the routine determination of HAV genome copies in shellfish specimens. The assay can be automated by using DNA enzyme immunoassay to save time and increase reliability and accuracy.

A new interface procedure has been developed that allows, for the first time, the high-efficiency analysis of synthetic oligonucleotides up to 75 bases by reversed-phase HPLC and on-line electrospray ionization mass spectrometry. For oligonucleotides up to 30 bases in length, single-base resolution can be obtained with low levels of cation adduct formation in the negative ion electrospray mass spectra. A key part of the method uses 1,1,1,3,3,3-hexafluoro-2-propanol as an additive to the HPLC mobile phase, adjusted to pH 7.0 with triethylamine. This novel additive results in both good HPLC separation and efficient electrospray ionization. The broad potential of this new method is demonstrated for synthetic homopolymers of thymidine (PolyT), fragments based on the pBR322 plasmid sequence, and phosphorothioate ester antisense oligonucleotides. This approach will be of particular utility for the characterization of DNA probes and PCR primers and quality control of antisense compounds such as phosphorothioates and their metabolites, as well as of materials used in clinical trials.

Nucleic acid sequence-based amplification (NASBA) has proved to be an ultrasensitive method for HIV-1 diagnosis in plasma even in the primary HIV infection stage. This technique was combined with fluorescence correlation spectroscopy (FCS) which enables online detection of the HIV-1 RNA molecules amplified by NASBA. A fluorescently labeled DNA probe at nanomolar concentration was introduced into the NASBA reaction mixture and hybridizing to a distinct sequence of the amplified RNA molecule. The specific hybridization and extension of this probe during amplification reaction, resulting in an increase of its diffusion time, was monitored online by FCS. As a consequence, after having reached a critical concentration of 0.1-1 nM (threshold for unaided FCS detection), the number of amplified RNA molecules in the further course of reaction could be determined. Evaluation of the hybridization/extension kinetics allowed an estimation of the initial HIV-1 RNA concentration that was present at the beginning of amplification. The value of initial HIV-1 RNA number enables discrimination between positive and false-positive samples (caused for instance by carryover contamination) - this possibility of discrimination is an essential necessity for all diagnostic methods using amplification systems (PCR as well as NASBA). Quantitation of HIV-1 RNA in plasma by combination of NASBA with FCS may also be useful in assessing the efficacy of anti-HIV agents, especially in the early infection stage when standard ELISA antibody tests often display negative results.

An immunoaffinity technique efficiently detects waterborne enteric viruses. The method involves indirect antibody capture of intact viruses and release of virion genomic RNA followed by reverse transcriptase PCR amplification and oligoprobe hybridization. The viruses are concentrated from huge volumes of water using 1MDS filters and washed with 1% beef extract-0.05 M glycine (BE/G). The BE/G eluate is concentrated and purified using polyethylene glycol (PEG) precipitation, ProCipitate precipitation, and then another PEG precipitation.

A combination of Pro-Cipitate precipitation and polyethylene glycol (PEG) concentration techniques efficiently purifies enteric viruses in oyster extracts for identification by reverse transcriptase-PCR amplification and oligoprobe. The acid-alcohol polymer, Pro-Cipitate, selectively precipitates proteins from nucleic acids, glycoproteins and oligosaccharides. Seeded viruses in the oyster extracts are purified by fluorocarbon extraction and concentrated using PEG. The method is quick and sensitive and efficiently detects Norwalk virus even at low concentrations.

The use of the innovative automated DNA synthesizer may yield a novel and effective approach in the incorporation of polypyridine metal complexes into synthetic oligonucleotides. Since the construction of oligonucleotide-metal conjugates can be done by using the new approach and the automated DNA synthesizer, metal centers can be easily determined. The photophysical processes of oligosaccharides can also be analyzed.

A four stranded motif of DNA conformations has been identified in the crystal structure of two distinct oligonucleotides. The four stranded motif, which is called the 'bi-loop,' has been identified in the linear heptamer and cyclic octamer that adopt similar structures. Using the nuclear magnetic resonance method, the dimeric 'bi-loop' structure in both sequence of oligonucleotides has been found in equilibrium with a monomeric dumbbell form.

An antigene triplex-forming oligonucleotide (TFO) strategy was devised to inhibit the expression of the HER-2/neu gene by preventing RNA polymerase elongation through the coding sequence of the HER-2/neu gene. Through in vitro transcription analysis in the presence and absence of triplex formation, an unmodified DNA TFO was found to be incapable of inhibiting RNA polymerase elongation. Triplex formation by an oligonucleotide-psoralen conjugate was employed to create a covalent photoadduct with a thymine on the nontemplate strand of the HER-2/neu gene. The results are discussed.