Frequency and distance of transposition of a modified Dissociation element in transgenic tobacco

Effective transposon tagging with the Ac/Ds system in heterologous plant species relies on the accomplishment of a potentially high transposon-induced mutation frequency. The primary parameters that determine the mutation frequency include the transposition frequency and the transposition distance. In addition, the development of a generally applicable transposon tagging strategy requires predictable transposition behaviour. We systematically analysed Ds transposition frequencies and Ds transposition distances in tobacco. An artificial Ds element was engineered with reporter genes that allowed transposon excision and integration to be monitored visually. To analyse the variability of Ds transposition between different tobacco lines, eight single copy T-DNA transformants were selected. For trans-activation of the Ds elements, different Ac lines were used carrying an unmodified Ac+ element, an immobilized sAc element and a stable Ac element under the control of a heterologous chalcone synthase (chsA) promoter. With all Ac elements, each Ds line showed characteristic and heritable variegation patterns at the seedling level. Similar Ds line-specificity was observed for the frequency by which Ds transpositions were germinally transmitted, as well as for the distances of the Ds transpositions. The sac element induced transposition of Ds late in plant development, resulting in low germinal transposition frequencies (0.37%) and high incidences of independent transposition (83%). The majority of these Ds elements (58%) transposed to genetically closed linked sites (≤10 cM)

Mutations at the Asc locus of tomato confer resistance to the fungal pathogen Alternaria alternata f. sp. lycopersici

The fungal pathogen Alternaria alternata f. sp. lycopersici produces host-selective AAL-toxins that cause Alternaria stem canker in tomato. Susceptibility to the disease is based on the relative sensitivity of the host to the AAL-toxins and is controlled by the Asc locus on chromosome 3L. Chemical mutagenesis was employed to study the genetic basis of sensitivity to AAL-toxins and susceptibility to fungal infection. Following the treatment of seeds of a susceptible line with ethyl methanesulphonate (EMS), resistant M2 mutants were obtained. Most plants with induced resistances showed toxin-sensitivity responses that were comparable to those of resistant control lines carrying the Asc locus. In addition, genetic analysis of the mutagenised plants indicated that the mutations occurred at the Asc locus. Furthermore, novel mutants were identified that were insensitive to the AAL-toxins at the seedling stage but toxin-sensitive and susceptible to fungal infection at mature stages. No AAL-toxin-insensitive insertion mutants were identified following a transposon mutagenesis procedure. Molecular mechanisms involved in host defence against A. a. lycopersici are discussed.

Molecular genetic characterisation of the Asc locus of tomato conferring resistance to the fungal pathogen Alternaria alternata f. sp. lycopersici

The Alternaria stem canker disease of tomato is caused by the fungal pathogen Alternaria alternata f. sp. lycopersici and its host-selective AAL-toxins. Resistance to the pathogen and insensitivity to the toxins are conferred by the Asc locus on chromosome 3L. Sensitivity to AAL-toxins is a relative character; the toxins inhibit development of all tested tomato tissues but susceptible cultivars are much more sensitive than resistant cultivars. In addition to tomato, some other plant and animal species are sensitive to the toxins as well. The likely mode of action of AAL-toxins is interference with sphingolipid biosynthesis by specific inhibition of ceramide synthase activity. To molecularly isolate Asc, transposon tagging and positional cloning strategies are applied. As a first step, transposon insertions and restriction fragment length polymorphism (RFLP) markers are identified in proximity of the Asc locus. Subsequently, the transposons are used to inactivate Asc by insertion mutagenesis, and the RFLP markers are used to identify yeast artificial chromosomes (YACs) with tomato DNA inserts. Once an Asc-insertion mutant and/or a YAC encompassing Asc has been obtained, physical isolation and characterisation of Asc will be conceivable. Elucidation of the molecular role of Asc will illuminate the specificity of host recognition by Alternaria alternata f. sp. lycopersici.

AGORA, a data- and biobank for birth defects and childhood cancer

BACKGROUND: Research regarding the etiology of birth defects and childhood cancer is essential to develop preventive measures, but often requires large study populations. Therefore, we established the AGORA data- and biobank in the Netherlands. In this study, we describe its rationale, design, and ongoing data collection. METHODS: Children diagnosed with and/or treated for a structural birth defect or childhood cancer and their parents are invited to participate in the AGORA data- and biobank. Controls are recruited through random sampling from municipal registries. The parents receive questionnaires about demographics, family and pregnancy history, health status, prescribed medication, lifestyle, and occupational exposures before and during the index pregnancy. In addition, blood or saliva is collected from children and parents, while medical records are reviewed for diagnostic information. RESULTS: So far, we have collected data from over 6,860 families (3,747 birth defects, 905 childhood cancers, and 2,208 controls). The types of birth defects vary widely and comprise malformations of the digestive, respiratory, and urogenital tracts as well as facial, cardiovascular, kidney, skeletal, and central nervous system anomalies. The most frequently occurring childhood cancer types are acute lymphatic leukemia, Hodgkin and non-Hodgkin lymphoma, Wilms' tumor, and brain and spinal cord tumors. Our genetic and/or epidemiologic studies have been focused on hypospadias, anorectal malformations, congenital anomalies of the kidney and urinary tract (CAKUT), and orofacial clefts. CONCLUSION: The large AGORA data- and biobank offers great opportunities for investigating genetic and nongenetic risk factors for disorders in children and is open to collaborative initiatives. Birth Defects Research (Part A) 106:675-684, 2016. (c) 2016 Wiley Periodicals, Inc