Agrobacterium and plant genetic engineering

Erratum

Agrobacterium and plant genetic engineering     

Influence of aerial dispersal on persistence and spread of pesticide-resistant Metaseiulus occidentalis in California almond orchards

Aerial dispersal of the phytoseiid Metaseiulus occidentalis (Nesbitt) was evaluated as a component in managing pesticide-resistant populations established in California almond orchards. Peak dispersal occurred in late July and early August during 1982 and 1983. Most predators (and spider mites) left the orchards on the prevailing winds from the northwest. Within the orchard, the prevailing winds had less influence, and dispersal was usually random. Both spider mites and predators dispersed randomly with regard to height from the almond trees, but data obtained during one 24-h interval suggest they do not disperse randomly throughout the day. Most aerial movements occurred between 16–22 h when relative humidity and wind speeds increased and temperatures decreased. Spider mites and predators were trapped on panels located 200 m from the orchard. A survey of carbaryl resistance levels in M. occidentalis collected from almond orchards surrounding the release sites indicates that carbaryl-resistant M. occidentalis dispersed at least 800 m between 1981–83. However, growers wishing to use the resistant strains should release them in their orchards as natural dispersal appears to be too slow. Migration of native M. occidentalis into the release sites appeared to be sufficiently rare that dilution of carbaryl-resistant populations was minimal during a 2–4 year period.

---

Résumé La dispersion aérienne du phytoseïdae, M. occidentalis (Nesbitt), a été estimée comme élément de la lutte contre les populations résistantes aux insecticides établies dans les vergers de Californie. La dispersion maximale s'est produite fin juillet et début a oût en 1982 et 1983. La plupart des prédateurs (et des acariens) quittent les vergers avec les vents dominants du nordouest. Dans le verger, les vents dominants sont moins importants et la dispersion est généralement au hasard. Tant les acariens que les prédateurs se dispersaient au hasard par rapport à la taille des amandiers, mais les relevés sur 24 heures laissent supposer qu'il n'y a pas une distribution aléatoire pendant la journée. La plupart des mouvements aériens se produisirent entre 16 et 22 heures quand HR et vitesse du vent augmentaient et température diminuait. Les acariens et prédateurs ont été piégés sur des panneaux à 200 m du verger.

     

Role of the spore coat layers in Bacillus subtilis spore resistance to hydrogen peroxide, artificial UV-C, UV-B, and solar UV radiation

Spores of Bacillus subtilis possess a thick protein coat that consists of an electron-dense outer coat layer and a lamellalike inner coat layer. The spore coat has been shown to confer resistance to lysozyme and other sporicidal substances. In this study, spore coat-defective mutants of B. subtilis (containing the gerE36 and/or cotE::cat mutation) were used to study the relative contributions of spore coat layers to spore resistance to hydrogen peroxide (H(2)O(2)) and various artificial and solar UV treatments. Spores of strains carrying mutations in gerE and/or cotE were very sensitive to lysozyme and to 5% H(2)O(2), as were chemically decoated spores of the wild-type parental strain. Spores of all coat-defective strains were as resistant to 254-nm UV-C radiation as wild-type spores were. Spores possessing the gerE36 mutation were significantly more sensitive to artificial UV-B and solar UV radiation than wild-type spores were. In contrast, spores of strains possessing the cotE::cat mutation were significantly more resistant to all of the UV treatments used than wild-type spores were. Spores of strains carrying both the gerE36 and cotE::cat mutations behaved like gerE36 mutant spores. Our results indicate that the spore coat, particularly the inner coat layer, plays a role in spore resistance to environmentally relevant UV wavelengths.     

Analysis of Fusarium causing dermal toxicosis in marram grass planters

In the European coastal dunes, marram grass (Ammophila arenaria) is planted in order to control sand erosion. In the years 1986 to 1991, workers on the Wadden islands in the Netherlands planting marram grass showed lesions of skin and mucous membranes, suggesting a toxic reaction. Fusarium culmorum dominated the mycoflora of those marram grass culms that were used for planting. This plant material had been cut and stored for more than one week in the open. The Fusarium toxin deoxynivalenol (DON) was detected in the suspect marram grass culms. Isolated F. culmorum strains were able to produce DON in vitro in liquid culture as well as in experimentally inoculated wheat heads. Pathogenicity tests, toxin test as well as RAPD analysis showed that the F. culmorum strains were not specialized for marram grass but may form part of the West-European F. culmorum population infecting cereals and grasses. Storage on old sand-dunes with plant debris may have led to the high occurrence of F. culmorum and contamination with DON. Marram grass culms should be obtained from young plantings on dunes on the seaward slopes and cut culms should not be stored.     

Refinement by linkage analysis in two large families of the candidate region of the third locus (SCA3) for autosomal dominant cerebellar ataxia type I

The autosomal dominant cerebellar ataxias (ADCA) are clinically and genetically heterogeneous. To date, several loci (SCAI-V) have been identified for ADCA type I. We have studied two large families from the northern part of The Netherlands with ADCA type I with a broad intra-familial variation of symptoms. In both families significant linkage is shown of the disease to the markers of the SCA3 locus on chromosome 14. Through recombinations, the candidate region for SCA3 could be refined to a 13-cM range between D14S256 and D14S81. No recombinations were detected with the markers D14S291 and D14S280, which suggests that the SCA3 gene lies close to these loci. This finding will benefit the individuals at risk in these two families who are seeking predictive testing or prenatal diagnosis.     

Comprehensive Ubiquitin E2 Profiling of Ten Ubiquitin E3 Ligases

The ubiquitin pathway regulates diverse functions including protein localization and stability. The complexity of the pathway involving nearly 40 identified E2 conjugating enzymes and over 600 E3 ligases raises the issue of specificity. With the E2s and E3s fitting into a limited number of classes based on bioinformatics, structures, and proven activities, there is not a clear picture as to what would determine which E2/E3 enzyme pair would be functional. There have been many reports of limited E2/E3 activity profiling with a small number of E2s and E3s. We have expanded on this to investigate the activity of ubiquitin E2s covering the majority of the reported classes/families in concert with a number of E3s implicated in a variety of diseases. Using an ELISA-based assay we screened 10 E3 ligases against a panel of 11 E2s to determine which E2/E3 pairs exhibited E3 autoubiquitylation activity. In addition, the ubiquitin chain linkage preference by certain E2/E3 pairs was investigated. Finally, substrate ubiquitylation was assayed for the E3 ligase MuRF1 using various E2/MuRF1 pairs. These studies demonstrate the utility of identifying the correct E2/E3 pair to monitor specific substrate ubiquitylation.     

In vitro characterisation of a monovalent and bivalent form of a fully human anti Ep-CAM phage antibody

 Antibodies to tumour-associated antigens are increasingly being used as targeting vehicles for the visualisation and for therapy of human solid tumours. The epithelial cell adhesion molecule (Ep-CAM) is an antigen that is overexpressed on a variety of human solid tumours and constitutes an attractive target for immunotargeting. We set out to obtain fully human antibodies to this antigen by selecting from a large antibody repertoire displayed on bacteriophages. Two single-chain variable antibody fragments (scFv) were identified that specifically bound recombinant antigen in vitro. One of the selected antibodies (VEL-1) cross-reacted with extracellular matrix components in immunohistochemistry of colon carcinoma, whereas the other scFv (VEL-2) specifically recognised colon cancer cells. The latter antibody was further characterised with respect to epitope specificity and kinetics of antigen-binding. It showed no competition with the well-characterised anti Ep-CAM MOC-31 monoclonal antibody and had an off-rate of 5 × 10⁻² s⁻¹. To obtain an antibody format more suitable for in vivo tumour targeting and to increase the apparent affinity through avidity, the genes of scFv VEL-2 were re-formatted by fusion to a human (γ1) hinge region and CH3 domain. This “minibody” was expressed in Escherichia coli, specifically bound the Ep-CAM antigen and showed a 20-fold reduced off-rate in surface plasmon resonance analysis. These results show that phage antibody selection, combined with antibody engineering, may result in fully human antibody molecules with promising characteristics for in vivo use in tumour targeting. Received: 13 July 2000 / Accepted: 12 October 2000