Effects of 2,4-Dichlorophenol, a Metabolite of a Genetically Engineered Bacterium, and 2,4-Dichlorophenoxyacetate on Some Microorganism-Mediated Ecological Processes in Soil

A genetically engineered microorganism, Pseudomonas putida PPO301(pRO103), and the plasmidless parent strain, PPO301, were added at approximately 10⁷ CFU/g of soil amended with 500 ppm of 2,4-dichlorophenoxyacetate (2,4-D) (500 μg/g). The degradation of 2,4-D and the accumulation of a single metabolite, identified by gas chromatography-mass spectrophotometry as 2,4-dichlorophenol (2,4-DCP), occurred only in soil inoculated with PPO301(pRO103), wherein 2,4-DCP accumulated to >70 ppm for 5 weeks and the concentration of 2,4-D was reduced to <100 ppm. Coincident with the accumulation of 2,4-DCP was a >400-fold decline in the numbers of fungal propagules and a marked reduction in the rate of CO₂ evolution, whereas 2,4-D did not depress either fungal propagules or respiration of the soil microbiota. 2,4-DCP did not appear to depress the numbers of total heterotrophic, sporeforming, or chitin-utilizing bacteria. In vitro and in situ assays conducted with 2,4-DCP and fungal isolates from the soil demonstrated that 2,4-DCP was toxic to fungal propagules at concentrations below those detected in the soil.     

Fanconi anemia: evidence for linkage heterogeneity on chromosome 20q

Fanconi anemia is a rare autosomal recessive disorder in which affected individuals are predisposed to acute myelogenous leukemia and other malignancies. We report the results of a genetic linkage study involving 34 families enrolled in the International Fanconi Anemia Registry. A significant lod score was obtained between D20S20, an anonymous DNA segment from chromosome 20q, and Fanconi anemia (Zmax 3.04, theta max = 0.12). However, six other anonymous DNA segments from chromosome 20q, including D20S19, which is highly polymorphic and tightly linked to D20S20, showed no or only weak evidence for linkage to Fanconi anemia. An admixture test revealed significant evidence for linkage heterogeneity (chi 2 = 6.10, P = 0.01) at the D20S19 locus. Lod scores suggestive of linkage between Fanconi anemia and this locus were obtained with two of the largest kindreds studied (lods = 2.6 and 2.1, at theta = 0.001). Thus, our data support the provisional assignment of a Fanconi anemia gene to chromosome 20q.     

Immunohistochemical identification of tubular segments in percutaneous renal biopsies

Summary To identify the renal cortical tubular segments involved in tubulo-interstitial disease in formalin-fixed, paraffin-embedded percutaneous kidney biopsies, we developed multiple immunolabeling protocols using segment-specific tubular markers. The present study of biopsies from patients with minimal change or thin basement membrane nephropathy provides a baseline for interpretation of histopathology. Proximal tubules were stained either by the PAS reaction or by the biotinylated Phaseolus vulgaris erythroagglutinin (PHA-E)-streptavidin-gold-silver system (brush borders black). The anti-Tamm-Horsfall (THP) antibody-immunoperoxidase (aminoethylcarbazole, AEC-IPO), and anti-epidermal cytokeratins (ECK) antibodies-immunoalkaline-Fast Blue BB methods marked the distal straight tubules and the cortical collecting system red-brown and blue, respectively. When these immunolabelings were combined, the coapplication of AEC-PO-labeled peanut agglutinin (PNA) or anti-epithelial membrane antigen antibody-AEC-IPO technique (both are markers for distal nephron) visualized the apical membranes of distal convoluted tubules. In the protocol PHA-E + PNA + THP + ECK, the tubular basement membranes were outlined by the anti-laminin antibody-AEC-IPO staining, carried out simultaneously. The protocol PNA + THP + ECK + PAS was found to be a quite appropriate multiple immunolabeling method for the tubules, and is recommended for use as a tool in the study of tubulo-interstitial diseases.Abbreviations PAS periodic acid-Schiff reaction - PHAE Phaseolus vulgaris erythroagglutinin - PNA Peanut agglutinin - EMA epithelial membrane antigen - THP Tamm-Horsfall glycoprotein - ECK epidermal cytokeratins - PO peroxidase - Biot-PHA-E biotinylated PHA-E - APAAP complexes of alkaline phosphatase and mouse monoclonal anti-alkaline phosphatase - SWARI swine anti-rabbit immunoglobulins - FCS fetal calf serum - TBS Tris-buffered saline - AEC aminoethylcarbazole - DAB diaminobenzidine - FBBB Fast Blue BB - IA immunoalkaline - GL glomerulus - PT proximal tubule - DST distal straight tubule - DCT distal convoluted tubule - CCS cortical collecting system - CT connecting tubule - CD collecting duct     

Inhibition of the restriction endonuclease BanII using modified DNA substrates. Determination of phosphate residues critical for the formation of an active enzyme-DNA complex

The restriction endonuclease BanII catalyzes the cleavage of double-stranded DNA and recognizes the degenerate sequence 5'-GPuGCPyC-3'. The poly-linker of M13mp18 contains one such sequence, 5'-GAGCTC-3'. The three other possible sites recognized by the enzyme were prepared by site-directed muta-genesis. The substitution of phosphate groups by phosphorothioate residues at some positions within the various recognition sites had relatively little effect on the rate of cleavage of the DNA. However, when the DNA contained a phosphorothioate group at the site of cleavage the rate of linearization of the DNA was decreased by a factor of 9. Interestingly, DNA which contained an additional phosphorothioate internucleotidic linkage immediately 3'-outside the recognition site could not be linearized by the enzyme. The results indicate that an important contact between enzyme and substrate is perturbed by the presence of the sulfur atom at this position.     

Carbon-13 NMR relaxation studies of pre-melt structural dynamics in [4-13C-uracil] labeled E. coli transfer RNAIVal.

We report 67.8 MHz carbon-13 spin-lattice relaxation studies on [4-13C-uracil] labeled tRNAIVal purified from E. coli SO-187. Following 13C-enriched C4 carbonyl resonances from modified and unsubstituted uridines scattered throughout the polymer backbone enables us to determine dynamical features in both loop and helical stem regions. The experimental results have been analyzed in terms of a model of isotropic overall molecular reorientation. "Anomalous" residues for which the experimental data cannot be accounted for in terms of the model provide an assessment of local and regional properties. Thus, "native" tRNAIVal under physiological conditions of magnesium (10 mM) and temperature (20 degrees - 40 degrees C), exhibits the following characteristics: 1) uridines held rigidly in helical stems and tertiary interactions display correlation times for rotational reorientation of 15-20 nsecs, typical for overall tRNA motion; 2) uridines in loops such as the wobble residue uridine-5-oxyacetic acid (V34) are quite accessible to solvent; moreover V34 and another loop residue, D17, exhibit local mobility; 3) the tertiary interactions involving 4-thio uridine (s4U8) and A14 and ribothymidine (rT54) and A58 are weakened as temperature increases.     

Specificity in the inactivation of enzymes by periodate-oxidized nucleotides

Periodate-oxidized ATP (oATP) inactivates the partial reaction of aminoacyl-tRNA synthetases in which amino acid is transferred to tRNA without altering the other partial reaction in which ATP is a substrate or a product. The inactivation has been shown to be nonspecific with regard to substituents on the dialdehyde and with regard to the enzymes susceptible to inactivation; oxidized GTP and oxidized uridine react as well as oATP with aminoacyl-tRNA synthetases and all three dialdehydes also inactivate rabbit muscle aldolase.