Disruption, replacement, and cosuppression of nitrate assimilation genes in Stagonospora nodorum.

We used Stagonospora (Septoria) nodorum to explore gene disruption as a general method of fungicide target validation. Nitrate reductase was chosen as a model target because the gene (NIA1) has been cloned from S. nodorum and disruptants should have a readily detectable phenotype (chlorate resistant and nitrate nonutilizing). We have succeeded in disrupting the NIA1 gene by both integration of an unselected vector during cotransformation and one-step gene replacement. Around 2% of transformants from the cotransformation approach became nitrate nonutilizing and Southern analysis confirmed disruption of the resident NIA1 gene. Half of the transformants with the gene replacement vector showed the nitrate nonutilizing phenotype expected from disruption. However, Southern analyses of 14 of these transformants showed that only 6 contained the expected NIA1 gene replacement. Of the remaining transformants, 6 had integrated multiple copies of the vector elsewhere in their genome and still had a functional nitrate reductase gene. Their inability to utilize nitrate was due to a lack of nitrite reductase activity. How this phenotype arose is not clear, but it might involve cosuppression of the nitrite reductase gene as the vector carried 1. 1 kb of the coding region and the complete 5' region of this gene which is adjacent to NIA1. Mutants of both types retained full pathogenicity in detached leaf assays, thereby invalidating both nitrate and nitrite reductase as fungicide targets.

Contrast media induced changes in tubular electrophysiology and glomerular filtration rate in the mouse kidney.

The isolated perfused mouse proximal tubule was used to examine electrophysiologic effects of diatrizoate and ioversol. Luminal or basolateral application of diatrizoate resulted in a dose-dependent, reversible hyperpolarization of the proximal tubule cell basolateral membrane potential (VB), which could be abolished by the addition of 10 microM probenecid. While there was a modest reduction in intracellular ATP following a 60-min exposure to diatrizoate, there was no deterioration of VB after 90 min of diatrizoate exposure, even following a 20-min hypoxic insult. Ioversol did not elicit an electrical response. Neither diatrizoate nor ioversol significantly affected transepithelial potential (VT) in the isolated perfused medullary thick ascending limb. In vivo studies showed that only the ionic contrast agent diatrizoate significantly reduced glomerular filtration rate, by 70%. The observed acute contrast media induced reduction in glomerular filtration rate does not appear to depend on direct renal tubular cytotoxicity.

Characterization of chitin synthase from Botrytis cinerea.

Chitin synthase in a microsomal preparation from Botrytis cinerea had an apparent Km for UDP-N-acetylglucosamine of 2.0 mM while nikkomycin Z and polyoxin D inhibited enzyme activity competitively with apparent Ki values of approximately 0.1 microM and 6 microM respectively. The organophosphorus fungicide edifenphos was a non-competitive inhibitor (Ki(app) 54 microM). Preincubation of microsomes for 2 h at 25 degrees C resulted in a maximum twofold stimulation of chitin synthase activity while preincubation with trypsin (25 micrograms ml-1) or cytosol (350 micrograms cytosolic protein ml-1) for 10 min at 25 degrees C resulted in approximately fourfold and 20-fold increases in chitin synthase activity, respectively. A range of protease inhibitors reduced the degree of activation of microsomal chitin synthase by cytosol. Most potent were phenylmethanesulphonyl fluoride and chymostatin; these compounds completely inhibited activation of enzyme activity. Two fragments (approx. 600 bp; CHS1 and CHS2) were amplified from B. cinerea genomic DNA using degenerate PCR primers based on regions of complete amino acid homology between previously published chitin synthase gene sequences. When the DNA and predicted amino acid sequences of CHS1 were used to probe computer databases for related sequences, B. cinerea CHS1 was found to be most similar to CHS1 from Neurospora crassa.

Flow velocity profile via time-domain correlation: error analysis and computer simulation.

An ultrasonic human-blood-flow velocity profile measurement method using time-domain correlation of consecutive echo pairs has been developed. The time shift between a pair of range gated echoes is estimated by searching for the shift that results in the maximum correlation. The time shift indicates the distance a group of scatterers has moved, from which flow velocity is estimated. The basis for the computer simulations and error analyses of the scheme includes a band-passed white Gaussian noise signal model for an echo from a scattering medium, the estimate of flow velocity from both a single scatterer and multiple scatterers, and a derived precision estimation. The error analysis via computer simulation includes an evaluation of errors associated with the correlation method. For a uniform flow velocity profile, beamwidth modulation represents the greatest error source. However, for a nonuniform flow velocity profile, the jitter caused by a small flow velocity gradient can exceed the other error sources. A detailed computer simulation evaluated the interdependencies of window length, beam width, vessel diameter, and viewing angle on the estimation of flow velocity.

Physical and genetic analysis of IS110, a transposable element of Streptomyces coelicolor A3(2).

On at least three independent occasions a 1.6 kb segment of Streptomyces coelicolor DNA was detected in apparently the same location in an attP-deleted derivative of the temperate phage phiC31 that carried a selectable viomycin resistance gene. This sequence (termed IS110) allowed integration of the phage (giving viomycin-resistant transductants) at homologous sequences (detected by Southern hybridisation) at several locations in the S. coelicolor genome. The inserted prophages facilitated genetic mapping of two IS110 copies in the chromosomal linkage map. A third copy did not exhibit simple segregation with chromosomal markers, and there appeared to be a frequent DNA rearrangement close to this copy. Some variation in the number of copies of IS110 and their location has taken place in the pedigree of S. coelicolor derivatives. IS110 did not hybridise to any known S. coelicolor plasmid, nor to any of several other IS-like elements previously described in other Streptomyces plasmids or phages. It hybridised strongly to DNA from only a small minority of other Streptomyces species and was absent from S. lividans, a close relative of S. coelicolor.