Encoded metal nanoparticle-based molecular beacons for multiplexed detection of DNA

In this paper we describe a molecular beacon format assay in which encoded nanowire particles are used to achieve multiplexing. We demonstrate this principle with the detection of five viral pathogens; Hepatitis A virus, Hepatitis C virus, West Nile Virus, Human Immune Deficiency virus and Severe Acute Respiratory Syndrome virus. Oligonucleotides are designed complementary to a target sequence of interest containing a 3′ universal fluorescence dye. A 5′ thiol causes the oligonucleotides to self-assemble onto the metal nanowire. The single-stranded oligonucleotide contains a self-complementary hairpin stem sequence of 10 bases that forces the 3′ fluorophore to come into contact with the metallic nanowire surface, thereby quenching the fluorescence. Upon addition of target DNA, there is hybridization with the complementary oligonucleotides. The resulting DNA hybrid is rigid, unfolds the hairpin structure, and causes the fluorophore to be moved away from the surface such that it is no longer quenched. By using differently encoded nanowires, each conjugated with a different oligonucleotide sequence, multiplexed DNA assays are possible using a single fluorophore, from a multiplexed RT-PCR reaction.     

pH-Induced Molten Globule State of <Emphasis Type="Italic">Rhizopus niveus</Emphasis> Lipase is More Resistant Against Thermal and Chemical Denaturation Than Its Native State

Here, we have characterized four pH-dependent states: alkaline state, “B” (pH 9.0), native state, “N” (pH 7.4), acid-induced state, “A” (pH 2.2) and molten globule state, “MG” (pH 1.8) of Rhizopus niveus lipase (RNL) by CD, tryptophanyl fluorescence, ANS binding, DLS, and enzyme activity assay. This “MG” state lacks catalytic activity and tertiary structure but it has native-like significant secondary structure. The “R _h” of all the four states of RNL obtained from DLS study suggests that the molecular compactness of the protein increases as the pH of solution decreases. Kinetic analysis of RNL shows that it has maximum catalytic efficiency at state “B” which is 15-fold higher than state “N.” The CD and tryptophanyl fluorescence studies of RNL on GuHCl and temperature-induced unfolding reveal that the “MG” state is more stable than the other states. The DSC endotherms of RNL obtained at pH 9.0, 7.4, and 2.2 were with two transitions, while at pH 1.8 it showed only a single transition.     

Fungal bioconversion of toxic polychlorinated biphenyls by white-rot fungus, Phlebia brevispora

Toxic coplanar polychlorinated biphenyls (Co-PCBs) were used as substrates for a degradation experiment with white-rot fungus, Phlebia brevispora TMIC33929, which is capable of degrading polychlorinated dibenzo-p-dioxins. Eleven PCB congener mixtures (7 mono-ortho- and 4 non-ortho-PCBs) were added to the cultures of P. brevispora and monitored by high resolution gas chromatography and mass spectrometry (HRGC/HRMS). Five PCB congeners, 3,3′,4,4′-tetrachlorobiphenyl, 2,3,3′,4,4′-pentachlorobiphenyl, 2,3′,4,4′,5-pentachlorobiphenyl, 3,3′,4,4′,5-pentachlorobiphenyl, and 2,3′,4,4′,5,5′-hexachlorobiphenyl were degraded by P. brevispora. To investigate the fungal metabolism of PCB, each Co-PCB was treated separately by P. brevispora and the metabolites were analyzed by gas chromatography and mass spectrometry (GC/MS) and identified on the basis of the GC/MS comparison with the authentic compound. Meta-methoxylated metabolite was detected from the culture containing each compound. Additionally, para-dechlorinated and -methoxylated metabolite was also detected from the culture with 2,3,3′,4,4′-pentachlorobiphenyl, 2,3′,4,4′,5-pentachlorobiphenyl, and 2,3′,4,4′,5,5′-hexachlorobiphenyl, which are mono-ortho-PCBs. In this paper, we identified the congener specific degradation of coplanar PCBs by P. brevispora, and clearly proved for the first time by identifying the metabolites that the white-rot fungus, P. brevispora, transformed recalcitrant coplanar PCBs.     

Hydrostatic pressure induces the fusion-active state of enveloped viruses.

Enveloped animal viruses must undergo membrane fusion to deliver their genome into the host cell. We demonstrate that high pressure inactivates two membrane-enveloped viruses, influenza and Sindbis, by trapping the particles in a fusion-intermediate state. The pressure-induced conformational changes in Sindbis and influenza viruses were followed using intrinsic and extrinsic fluorescence spectroscopy, circular dichroism, and fusion, plaque, and hemagglutination assays. Influenza virus subjected to pressure exposes hydrophobic domains as determined by tryptophan fluorescence and by the binding of bis-8-anilino-1-naphthalenesulfonate, a well established marker of the fusogenic state in influenza virus. Pressure also produced an increase in the fusion activity at neutral pH as monitored by fluorescence resonance energy transfer using lipid vesicles labeled with fluorescence probes. Sindbis virus also underwent conformational changes induced by pressure similar to those in influenza virus, and the increase in fusion activity was followed by pyrene excimer fluorescence of the metabolically labeled virus particles. Overall we show that pressure elicits subtle changes in the whole structure of the enveloped viruses triggering a conformational change that is similar to the change triggered by low pH. Our data strengthen the hypothesis that the native conformation of fusion proteins is metastable, and a cycle of pressure leads to a final state, the fusion-active state, of smaller volume.     

Abundant conserved microRNA target sites in the 5′-untranslated region and coding sequence

Recent studies have shown that miRNAs can target the promoter and CDS region. Thus, we predicted miRNA target sites in the 5′-UTR, CDS and 3′-UTR of Homo sapiens, Mus musculus and Drosophila melanogaster using miRanda and TargetScan. Target-site densities normalized with the average region length were higher in the 5′-UTR than 3′-UTR in all three organisms but were lower in the negative data set. Interestingly, the putative target sites were more conserved than non-target regions in both the 5′-UTR and 3′-UTR, implying that target sites in the 5′-UTR are subject to high selective pressure and might be functional. In Drosophila, 48 of 78 (61.5%) miRNAs showed high similarities with predicted siRNAs. Based on the results of previous experimental studies and a large-scale statistical analysis, we conclude that miRNA-mediated regulation is not limited to the 3′-UTR. However, the functionality of target sites in the 5′-UTR and CDS requires thorough investigation.     

Proton Motive Force in <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> Under Anaerobic Conditions in the Dark

In order to examine the mediatory role of proton motive force (∆p) or proton ATPase in H₂ production by Rhodobacter sphaeroides, ∆p was determined under anaerobic conditions in the dark, and the ATPase activity has been studied in R. sphaeroides strain A-10, isolated from Arzni mineral springs in Armenia. Membrane potential (∆φ) was measured from the distribution of tetraphenylphosphonium cation; pH gradient (∆pH) was the difference between the external and cytoplasmic pH values, and the latter was measured by 9-aminoacridine (9-AA) fluorescence changes. At pH 7.5, ∆φ was of −94 mV and the reversed ∆pH was +30 mV, resulting in ∆p of −64 mV. The addition of N,N′-dicyclohexylcarbodiimide (DCCD), the F₀F₁–ATPase inhibitor, was not affect ∆φ. It was shown that ∆φ varies nearly linearly with ΔpH, ∆φ increased from −57.1 mV at pH 6.0 to −103.8 mV at pH 8.0; it was compensated at high external pH by a reversed ∆pH, resulting in a low ∆p under anaerobic-dark conditions. Intracellular ATP concentrations and energetic charge (EC) were measured to evaluate a metabolism activity of R. sphaeroides.     

The New River, Virginia, muskellunge fishery: population dynamics, harvest regulation modeling, and angler attitudes

Although muskellunge, Esox masquinongy, fisheries in northern US states and Canadian provinces are increasingly being managed by introduction of restrictive harvest regulations (e.g. 1370-mm (54′′) minimum length limits), many southern US muskellunge fisheries continue to be managed with comparatively liberal regulations (e.g. 762-mm (30′′) minimum length limits) that are implemented statewide. We studied the population dynamics of the New River, Virginia, muskellunge fishery and used predictive modeling to determine whether restrictive harvest regulations also might prove beneficial for this southern latitude fishery. A creel survey was also conducted to learn more about angler attitudes to the New River muskellunge fishery. Muskellunge grew quickly, with fish reaching harvestable lengths (762 mm, 30′′) in 2–3 years. Muskellunge fishing pressure, harvest rates, and voluntary release rates were low compared with reports for more northern areas. Most anglers, irrespective of how often they fished for muskellunge, defined “trophy” muskellunge to be approximately 1050–1100 mm (41–43′′) in length. Although angler support for restrictive harvest regulations was low, abundance of memorable-length (≥1070 mm, 42′′) muskellunge was predicted to increase under all evaluated length limits. Muskellunge yield would remain static at 914-mm (36′′) and 1016-mm (40′′) length limits, because of the rapid growth of fish, but yield would decline dramatically with a 1143-mm (45′′) length limit, because male muskellunge rarely exceeded 1100 mm (43′′). Because of rapid growth and low release rates, implementation of higher length limits (e.g. 965–1067 mm, 38–42′′) may indeed prove beneficial for augmenting “trophy” muskellunge production on the New River. Angler support for higher minimum length limits might be increased by educating anglers about the rapid growth rates of muskellunge and the expected size structure changes that will result from a length-limit increase. Size structure changes resulting from an increase in the minimum length limit may be difficult to detect because of potential increases in fishing pressure or reduced fish growth as a result of competition for food resources. Long-term monitoring of muskellunge growth and angling pressure may therefore be needed to ensure that new regulations are indeed benefitting the fishery.     

Fluorescence imaging and targeted distribution of bacterial magnetic particles in nude mice

Bacterial magnetic particles (BMPs) are of interest as potential carriers of bioactive macromolecules, drugs, or liposomes. In this study, a high-pressure homogenizer was used to disrupt Magnetospirillum gryphiswaldense strain MSR-1 cells, and BMPs were purified. BMPs were labeled with fluorescence reagent 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocianin perchlorate (DiI) and injected into the tail vein of BALB/c nude mice. Distribution of fluorescence signals of DiI–BMPs in vivo was examined using a whole-body fluorescence imaging system. The result showed that fluorescence signals were detected in liver, stomach, intestine, lungs, and spleen. However, transmission electron microscopy of ultrathin sections indicated that BMPs were mainly present in liver and lungs, but not in the other organs. BMPs could be useful as carriers for targeted drug therapy of diseases of the liver or lung.     

Swapped green algal promoters: aphVIII-based gene constructs with Chlamydomonas flanking sequences work as dominant selectable markers in Volvox and vice versa

Production of transgenic organisms is a well-established, versatile course of action in molecular biology. Genetic engineering often requires heterologous, dominant antibiotic resistance genes that have been used as selectable markers in many species. However, as heterologous 5′ and 3′ flanking sequences often result in very low expression rates, endogenous flanking sequences, especially promoters, are mostly required and are easily obtained in model organisms, but it is much more complicated and time-consuming to get appropriate sequences from less common organisms. In this paper, we show that aminoglycoside 3′-phosphotransferase gene (aphVIII) based constructs with 3′ and 5′ untranslated flanking sequences (including promoters) from the multicellular green alga Volvox work in the unicellular green alga Chlamydomonas and flanking sequences from Chlamydomonas work in Volvox, at least if a low expression rate is compensated by an enforced high gene dosage. This strategy might be useful for all investigators that intend to transform species in which genomic sequences are not available, but sequences from related organisms exist.     

19F NMR study on the biological Baeyer-Villiger oxidation of acetophenones

The biological Baeyer–Villiger oxidation of acetophenones was studied by ¹⁹F nuclear magnetic resonance (NMR). The ¹⁹F NMR method was used to characterise the time-dependent conversion of various fluorinated acetophenones in either whole cells of Pseudomonas fluorescens ACB or in incubations with purified 4′-hydroxyacetophenone monooxygenase (HAPMO). Whole cells of P. fluorescens ACB converted 4′-fluoroacetophenone to 4-fluorophenol and 4′-fluoro-2′-hydroxyacetophenone to 4-fluorocatechol without the accumulation of 4′-fluorophenyl acetates. In contrast to 4-fluorophenol, 4-fluorocatechol was further degraded as evidenced by the formation of stoichiometric amounts of fluoride anion. Purified HAPMO catalysed the strictly NADPH-dependent conversion of fluorinated acetophenones to fluorophenyl acetates. Incubations with HAPMO at pH 6 and 8 showed that the enzymatic Baeyer–Villiger oxidation occurred faster at pH 8 but that the phenyl acetates produced were better stabilised at pH 6. Quantum mechanical characteristics explained why 4′-fluoro-2′-hydroxyphenyl acetate was more sensitive to base-catalysed hydrolysis than 4′-fluorophenyl acetate. All together, ¹⁹F NMR proved to be a valid method to evaluate the biological conversion of ring-substituted acetophenones to the corresponding phenyl acetates, which can serve as valuable synthons for further production of industrially relevant chemicals. Journal of Industrial Microbiology & Biotechnology (2001) 26, 35–42. Received 20 April 2000/ Accepted in revised form 16 September 2000     

HIV-1 Integrase and Virus and Cell DNAs: Complex Formation and Perturbation by Inhibitors of Integration

HIV-1 integrase (IN) catalyzes integration of viral DNA into cell DNA through 3′-processing of viral DNA and strand transfer reactions. To learn on binding of IN to DNAs and IN inhibition we applied spectroscopy (circular dichroism, fluorescence) in a simplified model consisting in a peptide analogue (K156) of α4 helix involved in recognition of viral and cell DNA; an oligonucleotide corresponding to the U5′ LTR DNA end; and an inhibitor (TB11) of the diketo acid (DKA) family. Results extrapolated to IN show that: the enzyme binds viral DNA with high affinity and specificity, but cell DNA with low affinity and specificity; the affinity of TB11 for IN is high enough to impair the binding of IN to cell DNA, but not to viral DNA. This explains why TB11 is an inhibitor of strand transfer but not of 3′-processing. These results can help in the search of new IN inhibitors.     

Comparison of Amphibian and Human ClC-5: Similarity of Functional Properties and Inhibition by External pH

Loss of function mutations of the renal chloride channel, ClC-5, have been implicated in Dent's disease, a genetic disorder characterized by low weight proteinuria, hypercalciuria, nephrolithasis and, in some cases, eventual renal failure. Recently, our laboratory used an RT-PCR/RACE cloning strategy to isolate an amphibian cDNA from the renal epithelial cell line A6 that had high homology to human ClC-5. We now report a full-length native ClC-5 clone (xClC-5, containing 5′ and 3′ untranslated regions) isolated by screening a cDNA library from A6 cells that was successfully expressed in Xenopus oocytes. In addition, we compared the properties of xClC-5 and hClC-5 using isogenic constructs of xClC-5 and hClC-5 consisting of the open reading frame subcloned into an optimized Xenopus expression vector. Expression of the full-length ``native' xClC-5 clone resulted in large, strongly rectifying, outward currents that were not significantly affected by the chloride channel blockers DIDS, DPC, and 9AC. The anion conductivity sequence was NO⁻ ₃ > Cl⁻= I⁻ > HCO⁻ ₃ >> glutamate for xClC-5 and NO⁻ ₃ > Cl⁻ > HCO⁻ ₃ > I⁻ >> glutamate for hClC-5. Reduction of the extracellular pH (pH _o ) from 7.5 to 5.7 inhibited outward ClC-5 currents by 27 ± 9% for xClC-5 and 39 ± 7% for hClC-5. The results indicate that amphibian and mammalian ClC-5 have highly similar functional properties. Unlike hClC-5 and most other ClC channels, expression of xClC-5 in oocytes does not require the removal of its untranslated 5′ and 3′ regions. Acidic solutions inhibited both amphibian and human ClC-5 currents, opposite to the stimulatory effects of low external pH on other ClC channels, suggesting a possibly distinct regulatory mechanism for ClC-5 channels. Received: 28 August 1998/Revised: 13 January 1999     

Chloride Currents Activated by Calcitonin and cAMP in Primary Cultures of Rabbit Distal Convoluted Tubule

Chloride (Cl⁻) conductances were studied in primary cultures of the bright part of rabbit distal convoluted tubule (DCTb) by the whole cell patch clamp technique. The bath solution (33°C) contained (in mm): 140 NaCl, 1 CaCl₂, 10 N-2-hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4 and the pipette solution 140 N-methyl-d-glucamine (NMDG)-Cl, 5 MgATP, 1 ethylene-glycol-bis(b-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 HEPES, pH 7.4. We identified a Cl⁻ current activated by 10⁻⁵ m forskolin, 10⁻³ m 8-bromo adenosine 3′,5′-cyclic monophophosphate (8 Br-cAMP), 10⁻⁶ m phorbol 12-myristate 13-acetate (PMA), 10⁻³ m intracellular adenosine 3′,5′-cyclic monophophosphate (cAMP) and 10⁻⁷ m calcitonin. The current-voltage relationship was linear and the relative ion selectivity was Br⁻ > Cl⁻≫ I⁻ > glutamate. This current was inhibited by 10⁻³ m diphenylamine-2-carboxylate (DPC) and 10⁻⁴ m 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and was insensitive to 10⁻³ m 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). These characteristics are similar to those described for the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ conductance. In a few cases, forskolin and calcitonin induced an outwardly rectifying Cl⁻ current blocked by DIDS. To determine the exact location of the Cl⁻ conductance 6-methoxy-1-(3-sulfonatopropyl) quinolinium (SPQ) fluorescence experiments were carried out. Cultures seeded on collagen-coated permeable filters were loaded overnight with 5 mm SPQ and the emitted fluorescence analyzed by laser-scan cytometry. Cl⁻ removal from the apical solution induced a Cl⁻ efflux which was stimulated by 10⁻⁵ m forskolin, 10⁻⁷ calcitonin and inhibited by 10⁻⁵ m NPPB. In 140 mm NaBr, forskolin stimulated an apical Br⁻ influx through the Cl⁻ pathway. Forskolin and calcitonin had no effect on the basolateral Cl⁻ permeability. Thus in DCTb cultured cells, exposure to calcitonin activates a Cl⁻ conductance in the apical membrane through a cAMP-dependent mechanism. Received: 5 July 1995/Revised: 21 December 1995     

Cloning,expression, isolation,and properties of thymidine kinase from herpes simplex virus type 1, strain L2

Thymidine kinase UL23 gene (EC 2.7.1.145) from the L2 acyclovir-sensitive strain of herpes simplex virus type 1 was cloned and expressed in E. coli. The enzyme was purified by chromatography to the purity of 90% according to PAG electrophoresis data. The Michaelis constants for the reactions with thymidine and acyclovir were determined. The enzyme was found to phosphorylate modified nucleosides, particularly 3′-deoxythymidine, 3′-deoxy-2′,3′-didehydrothymidine, 2′,3′-dideoxycytidine, 9-[(hydroxyethyl)methyl]guanine, E-5-(2-bromovinyl-2′-deoxyuridine, 9-(1,3-dihydroxy-2-propoxymethyl)guanine, 2′,3′-dideoxydehydrothymidine, β-L-2′,3′-dideoxy-3′-thiacytidine, and 3′-fluoro-3′-deoxythymidine. Some properties of the purified enzyme were compared with those of thymidine kinases of other herpes simplex virus strains. It was shown that acyclovir H-phosphonate inhibited the enzyme.     

Mitochondrial sequestration of BCECF after ester loading in the giant alga Chara australis

Summary. Ratiometric fluorescent dyes are often used to monitor free ion concentrations in vivo, especially in cells that are recalcitrant to transformation with genetically encoded fluorescent markers. Although intracellular dye distributions are often found to be cytosolic, dye localisation has often not been examined in detail. We began exploring the use of BCECF (2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein) to monitor pH in the giant alga Chara australis and discovered that younger leaf cells could be loaded using the acetoxymethyl ester of BCECF. However, we were puzzled to find in microphotometric measurements that the fluorescence ratio appeared insensitive to manipulations affecting cytosolic pH. Confocal imaging of C. australis cells loaded with BCECF showed an accumulation of the dye in two locations: (1) on the outside of the chloroplasts in irregularly shaped stationary bodies; (2) within 1–1.5 μm structures that moved rapidly with the pericellular cytoplasmic streaming. Together with the streaming cytoplasm, these organelles were rendered stationary with 50 μM cytochalasin D. Rhodamine 123, a mitochondrionspecific dye, highlighted organelles outside of the chloroplasts, similar to those shown by BCECF in location 1. We conclude that in the cytoplasmic compartment, BCECF was sequestered within cytoplasmic mitochondria in immature and fast-growing cells and within the cortical mitochondrial system in older and slowly growing cells. Thus, BCECF-AM is unsuitable for reporting changes in cytosolic pH in C. australis but might be employed in future to study pH changes in the mitochondria. Correspondence: M. J. Beilby, Biophysics, School of Physics, University of New South Wales, Sydney 2052, Australia.     

Transformation of N ′,N ′-dimethyl-N -(hydroxyphenyl)ureas by laccase from the white rot fungus Trametes versicolor

Transformation of N′,N′-dimethyl-N-(hydroxyphenyl)ureas was assayed in the presence of purified laccase produced by the fungus Trametes versicolor. The para- and ortho-hydroxyphenyl derivatives were enzymatically transformed, whereas the meta derivative was not. The performance of laccase-mediated transformation depended on the pH, with an optimum for the para-derivative degradation rate at pH 5. The pH also influenced the nature of the reaction products. The chemical was exclusively oxidised into p-benzoquinone at pH 3 and into mainly N′,N′-dimethyl-N-[(2,5-cyclohexadiene-1-one)-4-ylidene]urea at pH 6. The ortho- derivative was transformed essentially into insoluble purple compounds, probably appearing as polymers resulting from coupling of the parent compound. Received: 14 September 1998 / Received revision: 23 November 1998 / Accepted: 29 November 1998     

In silico study of the inhibition of DNA polymerase by a novel catalpol derivative

In this work, a novel catalpol derivative (6,10,2′,6′-tetraacetyl-O-catalpol), which was previously obtained by our group and shown experimentally to inhibit a type of Taq DNA polymerase, was studied in silico. Studies of the interaction of 6,10,2′,6′-tetraacetyl-O-catalpol with the Klentaq fragment of the Taq DNA polymerase I from Thermus aquaticus helped to elucidate the mechanism of inhibition of the enzyme, and offered valuable information that can be used to propose substrate structural modifications aimed at increasing the binding affinity. Classical and semi-empirical methods were used to characterize the conformational preferences of this organic compound in solution. Using docking simulations, the most probable binding mode was found, and the stabilities of the docked solutions were tested in a series of molecular dynamics experiments. Results indicated that the mechanism of inhibition may be competitive, which agrees with previous binding experiments done with 6,10,2′,6′-tetraacetyl-O-catalpol.     

Influence of chroline substitution pattern on the degradation of polychlorinated biphenyls by eight bacterial strains

We compared the metabolism of eight di- and trichlorobiphenyls by eight bacterial strains chosen to represent a broad range of degradative activity against polychlorinated biphenyls (PCBs). The PCB congeners used were 2,3-, 2,3′-, 2,4′-, 3,3′-, 2,3,3′-, 2,4,4′-, 2,5,3′-, and 3,4,2′-chlorobiphenyl. The bacterial strains used wereCorynebacterium sp. MB1,Alcaligenes strainsA. eutrophus H850 andA. faecalis Pi434, andPseudomonas strains LB400 and H1130,P. testosteroni H430 and H336, andP. cepacia H201. The results indicated that both the relative rates of primary degradation of PCBs and the choice of the ring attacked were dependent on the bacterial strain used. The bacterial strains exhibited considerable differences in their relative reactivity preferences for attack on mono- and dichlorophenyl groups and in the degree to which the attack was affected by the chlorine substitution pattern on the nonreacting ring. For MB1 the reactivity pattern was 3-≥4-≫2-chlorophenyl with no attack on 2,4- or 2,5-chlorophenyl groups. This strain was relatively insensitive to the chlorine substitution pattern on the nonreacting ring. Strains H1130, H430, H201, and Pi434 exhibited the same reactivity preferences as MB1, but for these strains (and for all others tested) the chlorination pattern on the nonreacting ring had a strong effect. For strain H336 the reactivity preference was 4-≥2->2,4-≥3-chlorophenyl, with no evidence of attack on 2,5-chlorophenyl rings. For strains H850 and LB400 the relative reactivity was 2->2,5->3-≫2,4->4-chlorophenyl. On this basis we propose that the eight bacterial strains represent four distinct classes of biphenyl/PCB-dioxygenase activity. The types of products formed were largely strain-independent and were determined primarily by the chlorine substitution pattern on the reacting ring. When the reacting ring was an unsubstituted phenyl or a 2-chlorophenyl group, the products were chlorobenzoic acids in high yields; for a 3-chlorophenyl ring, both chlorobenzoic acids and chloroacetophenones in moderate yields; and for a 4- or 2,4-chlorophenyl group, chlorobenzoic acids in low yields with an apparent accumulation ofmeta ring-fission product. Strains H850 and LB400 were able to degrade the 3-chlorobenzoic acid that they produced from the degradation of 2,3′-chlorobiphenyl. We conclude that despite differences among strains in the specificity of the initial dioxygenase, the specificities of the enzymes responsible for the subsequent degradation to chlorobenzoic acid and/or chloroacetophenone are quite similar for all strains.