Investigation of interspecific genome-plastome incompatibility in Oenothera and Passiflora

Interspecific genome-plastome incompatibility is a widely observed phenomenon but its primary causes are still unknown. It reflects genome-plastome interactions that play a direct role in speciation processes, such interspecific combinations of nuclear genomes and plastomes that fail to develop fully autotrophic plants which then are usually eliminated by natural selection. We have investigated two plant models displaying genome-plastome incompatibility, Oenothera and Passiflora, using strategies of molecular biology in order to contribute to an analysis of primary causes of interspecific genome-plastome incompatibility. 1. Expressed sequence tags in Oenothera: In this study we present the first analyzed EST data set for Oenothera. 3,532 cDNA sequences derived from 9-week-old Oenothera plantlets were the analysed and assembled into 1,621 nonredundant clusters, including 1,133 singletons and 488 multi-member unigenes which contain a total of 875,940 nonredundand nucleotides. EST sequences were analysed by Sputnik algorithm. They were also used in the development of gene-specific PCR-based codominant markers (SNPs, CAPS, micro-satellites). The cDNA library could be directly used for macroarray applications including gene expression studies and for physical mapping. 2. Genotyping analyses in Oenothera using AFLP technology: The comparison of AFLPs from Oenothera with AFLPs from Arabidopsis was used to obtain an approximation of the genome size. The genotyping data provide evidence that genome of Oenothera is only six times larger than that of Arabidopsis corresponding to a size of about 750 Mb. The AFLP markers were also successfully applied to construct first genetic maps using F2 mapping population of interspecific hybrids between Oenothera elata ssp. hookeri, line johansen, AA-III, x Oenothera grandiflora ssp. tuscaloosa, BB-III. The linkage maps contain 88 AFLP markers covering a total map length of 154.4 cM for dominant markers in johansen, AA-III and 104 AFLP markers and a total size of 155.3 cM for dominant markers in grandiflora, BB-III. In addition, it was possible to assign genome-plastome incompatibility locus to the margin of coupling group 2B with 13 cM distance to the next AFLP marker. SUMMARY 91 The EST project followed by genotyping analysis increases knowledge and requirements in discovering primary causes of genome-plastome incompatibility. Oenothera with genome-plastome incompatibility, chromosomal translocations and many chromosomal arrangements provides an elegant tool in the study of genomeplastome interactions, speciation processes and species evolution. 3. Investigation of genome-plastome incompatibility in Passiflora: We present the first evidence of hybrid bleaching in this genus. The hybrid between Passiflora menispermifolia x Passiflora oerstedii showed bleaching regions during plant development. Reciprocal crosses have also shown hybrid bleaching but as well significant differences in leaf shape. Molecular analyses of cpDNA showed that Passiflora plastids are inherited bi-parentally and that the P. menispermifolia plastome is incompatible in F1 hybrids with P. oerstedii. This is the first evidence of genome-plastome incompatibility in Passiflora, which differ from Oenothera incompatibilities. The analysis of plastid ultrastructure showed that green tissues in the F1 generation have fully developed chloroplasts with thylakoids and grana; the incompatible material in F1 hybrids lacks differentiated plastids and contains plastids with only rudimentary membranes. An unexpected plastid ultrastructure was found in P. menispermifolia. The leaf from plant growing at greenhouse conditions contains plastids in different development stages including etioplasts, which normally develop from proplastids in darkness. Electron micrographs also indicated retardation of grana formation in P. menispermifolia which shows that vesicles could deliver parts of thylakoid components and that they may directly participate in the formation grana stacks. Northern and Western analyses demonstrated that genome-plastome incompatibility affects both transcription and translation, but with differences for nuclear and plastome encoded genes

Formal Verification throughout the Development of Robust Systems

As transistors are becomming smaller and smaller, they become more susceptible to transient faults due to radiation. A system can be modified to handle these faults and prevent errors that are visible from outside. We present a formal method for equivalence checking to verify that this modification does not change the nominal behavior of the system. On the other hand, we contribute an algorithm to formally verify that a circuit is robust against transient faults under all possible input assignments and variability. If equivalence or robustness cannot be shown, a counterexample is generated

The role of antibody in the killing of Pseudomonas aeruginosa by human sera

Antibody plays an important role in the protection against many different infections. Here the role of antibody in the protection against P aeruginosa infection was further investigated. Patients suffering from chronic lung infection with P. aeruginosa can produce antibodies that protect the bacterium from the bactericidal activity of serum. The effect of the removal of these antibodies on the P. aeruginosa population was investigated. One clonal lineage appeared to be eradicated from a patient with a multi-lineage infection. However, the treatment did not promote strain replacement in either patient suggesting that a single strain was able to persist within a specific niche in the lung. The role of antibody in the protection against P. aeruginosa infection by sera from healthy individuals was further investigated. Killing of P. aeruginosa was complement-dependent and in some instances, complement alone was sufficient to elicit killing. P. aeruginosa-specific antibodies enhanced the killing of some strains and blocked the killing of others. Inhibitory antibody was identified in healthy serum, suggesting that healthy individuals can produce inhibitory antibodies without an active infection. The mechanism of inhibitory antibodies is not fully understood. A transposon library was constructed in a strain of P. aeruginosa isolated from a non-cystic fibrosis bronchiectasis patient and used to define the essential genome. This was the first library to be constructed in a multi-drug resistant respiratory isolate and consisted of 577,494 unique mutants. The library can be used as a tool to provide further insight into the mechanism of inhibitory antibodies and has the potential to lead to the development of new treatments and diagnostics for patients with inhibitory antibody