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R P Oliver

Publications and source records attributed to R P Oliver.

10 recordsLinked to original sources

CfT-I: an LTR-retrotransposon in Cladosporium fulvum, a fungal pathogen of tomato.

A retrotransposon from the fungal tomato pathogen Cladosporium fulvum (syn. Fulvia fulva) has been isolated and characterised. It is 6968 bp in length and bounded by identical long terminal repeats of 427 bp; 5 bp target-site duplications were found. Putative first- and second-strand primer binding sites were identified. Three long open reading frames (ORFs) are predicted from the sequence. The first has homology to retroviral gag genes. The second includes sequences homologous to protease, reverse transcriptase, RNAse H and integrase, in that order. Sequence comparisons of the predicted ORFs indicate that this element is closely related to the gypsy class of LTR retrotransposons. Races of the pathogen exhibit polymorphisms in their complement of at least 25 copies of the sequence. Virus-like particles which co-sediment with reverse transcriptase activity were observed in homogenates of the fungus. This is the first report of an LTR retrotransposon in a filamentous fungus.

Amino Acid Sequence

Transformation frequencies are enhanced and vector DNA is targeted during retransformation of Leptosphaeria maculans, a fungal plant pathogen.

Leptosphaeria maculans, a fungal pathogen of Brassica spp., was successfully transformed with the vector pAN8-1, encoding phleomycin resistance. Protoplasts of a vigorous Phleor transformant were then retransformed using the partially homologous vector, pAN7-1 which encodes hygromycin B resistance. Retransformation of this strain to hygromycin resistance occurred at frequencies that were consistently twofold higher than with the original recipient strain. Linearised pAN7-1 DNA transformed phleomycin-resistant protoplasts at higher frequencies still. All the transformants that were tested retained a phleomycin-resistant phenotype (20/20). Molecular analysis of five transformants generated with circular pAN7-1 DNA indicated that in four cases the pAN7-1 vector had integrated into pAN8-1 sequences. These results suggest that transformation frequencies in L. maculans are limited by the ability of vector DNA to integrate into the genome. Hence, construction of strains with target sites for integration may prove to be a generally useful method for improving transformation frequencies of poorly characterised filamentous fungi, particularly when using heterologous vectors. This would greatly facilitate the identification of genes by transfer of gene libraries and the standardisation of chromosomal location effects in studies of expression of nested promoter deletions.

Ascomycota

Analysis of local electrogram characteristics correlated with successful radiofrequency catheter ablation of accessory atrioventricular pathways.

UNLABELLED: Due to the limited myocardial lesions produced by radiofrequency current, the ablation of accessory pathways (AP) requires precise localization of such connections. The purpose of this study was to ascertain which characteristic(s) of the local bipolar electrogram, recorded from the ablation and adjacent electrode immediately prior to the application of radiofrequency current, correlated with precision in localization adequate to permit AP ablation. Signal analysis was performed for 326 sets of electrograms preceding the attempted ablation of 107 APs in 100 consecutive patients. For 80 antegrade APs, the following variables were evaluated: (1) the presence or absence of an AP potential; (2) the local atrial-AP interval; (3) the local atrioventricular (AV) interval; and (4) the relationship between the onset of local ventricular depolarization and onset of delta wave of the surface electrocardiogram. For the 27 concealed APs, the following characteristics were evaluated: (1) the presence or absence of an AP potential; and (2) the local VA interval during reciprocating tachycardia or ventricular pacing. RESULTS: Antegrade APs: By statistical analysis, the best correlate of successful ablation of an antegrade AP was a local AV interval less than or equal to 40 msec (positive predictive value = 94%; 95% confidence intervals [CI] = 81%-100%). Local AV intervals less than or equal to 50 msec preceded 88% of successful AP ablations, compared to only 8% of failed radiofrequency current applications. The positive predictive value of the other variables were: presence of an AP potential: 35% (95% CI = 27%-40%); local atrial-AP intervals less than or equal to 40 msec: 54% (95% CI = 43%-66%); and local ventricular depolarization preceding onset of the delta wave 43% (95% CI = 34%-52%). For concealed APs, the positive predictive value of a VA interval less than 60 msec was 71% (95% CI = 48%-88%); the positive predictive value for the presence of an AP potential was 58% (95% CI = 32%-81%). CONCLUSIONS: No single electrogram characteristic had a positive predictive value and a sensitivity greater than 90% for AP localization adequate for radiofrequency current ablation. For antegrade APs, the best correlate of adequate localization was a local AV interval less than or equal to 40 msec; as a corollary, radiofrequency current applications at sites where the local AV was greater than 60 msec, were unlikely to be effective. Objective criteria for the localization of concealed APs were less certain. Electrogram analysis, as a guide to AP localization and ablation, requires careful analysis of multiple variables, with analysis of the local AV interval a salient objective factor.

Adult

Pulsed field gel electrophoresis reveals chromosome length differences between strains of Cladosporium fulvum (syn. Fulvia fulva).

Methods are described for the electrophoretic separation of chromosome-sized DNA molecules from the fungal tomato pathogen Cladosporium fulvum (syn. Fulvia fulva). Using a hexagonal electrode array and switching times of 75 min at 45 V for 14 days, nine bands could be resolved. By comparison with co-electrophoresed Aspergillus nidulans chromosomal DNA (which was resolved into seven bands), the sizes of the C. fulvum bands are estimated to be between 1.9 Mb and 5.4 Mb. The two largest bands are believed to be doublets, giving a minimum genome size of 44 Mb. Cloned probes for the ribosomal DNA repeat, an anonymous single copy fragment and a newly discovered retrotransposon were hybridized to blots of the pulsed field gels, demonstrating the use of this technique for genomic mapping. Most strains of C. fulvum had an identical pattern of bands. Two strains exhibited two polymorphisms which could be due to a translocation.

Blotting, Southern

The automatic implantable cardioverter-defibrillator in young patients.

An international survey identified 40 patients less than 20 years old who underwent surgical implantation of an automatic implantable cardioverter-defibrillator (AICD). There was a history of aborted sudden cardiac death or sustained ventricular tachycardia in 92.5% of these patients. Twenty-two patients (55%) had structural heart disease; dilated and hypertrophic cardiomyopathy were the most common diagnoses. Eighteen patients (45%) had primary electrical abnormalities including seven with the congenital long QT syndrome. There were no perioperative deaths associated with device implantation. Concomitant drug therapy was administered to 75% of the patients. Defibrillator discharge occurred in 70% of the patients, with 17 patients (42.5%) receiving at least one appropriate shock. There were two sudden and two nonsudden deaths at 28.2 months' median follow-up. Sudden death-free survival rates by life table analysis at 12 and 33 months were 0.94 and 0.88, respectively. Total survival rates at 12 and 33 months were 0.94 and 0.82, respectively. The AICD represents an effective treatment approach for young patients with life-threatening ventricular tachyarrhythmias.

Adolescent

Expression of the Escherichia coli beta-glucuronidase gene in industrial and phytopathogenic filamentous fungi.

A chimaeric beta-glucuronidase (GUS) gene has been created by ligating the Aspergillus nidulans glyceraldehyde 3-phosphate dehydrogenase promoter to the coding sequence of the E. coli uidA gene. Co-transformation of this vector into A. nidulans, A. niger and the tomato pathogen Fulvia fulva (syn. Cladosporium fulvum (Cooke] resulted in the expression of beta-glucuronidase. GUS activity was detected by growth on agar media containing X-gluc and by enzyme assays of mycelial extracts. Expression of the gene in F. fulva transformants was also easily detectable during growth in plants and did not affect pathogenicity. These results form the basis for a versatile and sensitive reporter gene system for industrial and phytopathogenic filamentous fungi.

Aspergillus nidulans

Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli.

A new, heterologous, dominant marker for selection of Aspergillus transformants is described. This marker is based on the Escherichia coli hygromycin B (HmB) phosphotransferase gene (hph). Expression of the hph gene is controlled by A. nidulans gpd and trpC expression signals. An Aspergillus transformation vector was constructed which contains this marker and confers HmB resistance to Aspergillus species. With both A. niger and A. nidulans, transformation frequencies of 5-20 transformants per micrograms vector DNA were obtained. Cotransformation with other vectors was shown to be very efficient in both species, when selection for HmB resistance was applied.

Anti-Bacterial Agents

Scabies.

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Humans

Expression of reverse transcriptase genes in Fulvia fulva.

Antibodies raised against intercellular fluid antigens isolated from diseased tomato leaves have revealed that the fungal pathogen Fulvia fulva expresses genes for a fungal reverse transcriptase (RNA-dependent DNA polymerase). This enzyme is required for the replication of retroviruses and retroviral-like transposable elements and could provide a mechanism for increasing the mutation rate of fungal pathogens, perhaps explaining their ability to evolve new races rapidly. We report here the DNA sequence of a 225-bp clone from a lambda gt11 genomic library of F. fulva. This clone, designated P5, exhibits a high degree of sequence homology with the reverse transcriptase (pol) gene of the Drosophila melanogaster copia-like retrotransposon 17.6. Southern blot analysis of genomic DNA of F. fulva showed that P5-related sequences are moderately reiterated with 30-100 copies, some of which exhibit restriction fragment length polymorphism in different races of the pathogen. Western blot analysis of extracts from F. fulva with antibodies raised to purified reverse transcriptase (from human immunodeficiency virus-1) revealed immunoreactive proteins. Reverse transcriptase previously has been detected in a variety of organisms including yeast, insects, protozoa, and mammals, but to our knowledge, this is the first report of its occurrence in filamentous fungi.

Amino Acid Sequence