Browsing by Subject "DNS"

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Bedeutung der c-Abl-Aktivität für die Reaktion auf DNA-Schädigung und für die genetische Stabilität Bcr-Abl-negativer Zellen
(2011) Fanta, Silke; Aulitzky, Walter E.
The launch of Imatinib (Glivec®, Gleevec®, STI571) in August 2001 was an important advancement in the therapy of chronic myeloid leukemia (CML). The small-molecule inhibitor directly targets the oncogenic tyrosine kinase Bcr-Abl, which has been identified as the central cause for the development of CML. Treatment with Imatinib is the gold standard in the therapy of CML. However, taking the current state of research, an elimination of the malignant Bcr Abl-positive clone cannot be achieved by treatment with Imatinib. Thus, long-term or even lifelong treatment of patients is necessary. As a consequence, it is of great interest to clarify the biological effects of Imatinib on physiologically normal cells. Previous studies of the group showed that Imatinib treatment of Bcr Abl-positive cells leads to a decreased mutation frequency following DNA damage. Within the scope of the present work, evidence for significantly enhanced mutation rates after DNA damage in non-cancerogenic primary human lymphocytes (PBMC) and murine hematopoietic cell lines (32D and BaF3) after Imatinib treatment was obtained for the first time. Thus, Imatinib treatment of Bcr Abl-negative cells shows opposite effect compared to Bcr Abl-positive cells. It was therefore proven that the Imatinib-related inhibition of Bcr Abl as well as the off-target effects in Bcr Abl-negative cells play an important role in the genetic stability. To determine whether an Imatinib-mediated inhibition of c Abl activity is responsible for effects independent of Bcr Abl, genetic c Abl models were used to assess stress-induced mutation frequency. To this, we employed c Abl-knockout-MEFs (embryonic mouse fibroblasts), which were retransfected with wild type c Abl and a kinase-deficient form, respectively. After DNA damage, there was a significant increase in mutation frequency in the kinase-deficient cells (MEF Abl-KD) when compared to the c-Abl wild type (MEF Abl-wt) cells. Consequently, c-Abl activity is of great importance for the maintenance of genetic stability. Several factors can result in an increased mutation frequency in cells. Examples include altered cell proliferation, impaired DNA repair mechanisms or a delayed induction of cell death. In the latter case, DNA damage is not adequately repaired and passed to the daughter cells. In this study, different hematopoietic cell lines were used to show that neither the pharmacological nor the genetic inhibition of c-Abl activity has an influence on induction of cell death, division rate, cloning efficiency and cell cycle distribution. To investigate how far Imatinib influences the kinetics of DNA strand break repair after irradiation, alkaline comet assays were performed. Imatinib treatment of cells had no influence on induction of strand breaks or constitutive strand breaks prior to irradiation. However, cells treated with Imatinib exhibited a significantly delayed repair of DNA strand breaks. This delay was shown in the same manner in hematopoietic cell line models and in primary human lymphocytes, which were treated with Imatinib as well as with Dasatinib, a second generation Abl-inhibitor. Cell line models with different forms of c-Abl were used to provide evidence that this effect is caused by inhibition of the c-Abl kinase activity. The delayed repair of DNA strand breaks was also seen in cells with a kinase-deficient form of c-Abl (MEF Abl KD). But treatment with Imatinib had no effect on the kinetics of DNA repair in cells that expressed an Imatinib-resistant form of c Abl (c Abl T315I). Double- (DSB) as well as single-strand breaks (SSB) are determined in an alkaline comet assay. By applying neutral conditions, this assay can be modified to exclusively analyze DSB repair. As expected, there was a significantly lower induction of DSB after irradiation when compared to the occurrence of SSB. However, Imatinib did neither influence the induction nor the kinetics of DSB repair. Both pulsed-field gel electrophoresis and the quantification of gamma-H2AX were used to confirm that Imatinib does not affect DSB repair. Rather, the delayed repair kinetics are exclusively caused by an Imatinib-dependent interference with SSB repair. Extensive investigations of the molecular signaling pathways of DNA damage repair show that inhibition of c Abl activity does not affect ATM-Chk2-p53 or ATR-Chk1 signaling. Poly(ADP-ribosyl)ation of proteins is an early event in the processing of the SSB repair. This modification of proteins by addition of long and branched poly(ADP-ribose) chains (PAR) is an essential part of the SSB repair and base excition repair (BER). Both the synthesis and the cleavage of PAR is mediated by the kinases PARP-1 (poly(ADP-ribose) polymerase-1) and PARG (poly(ADP-ribose) glycohydrolase). This activity was determined by quantification of PAR and the percentage of cells, which were PAR-positive at a certain time. Possible effects of an Imatinib-induced inhibtion of c-Abl on poly(ADP-ribosyl)ation were investigated. To this, a method for the measurement of PAR events on a single-cell level was established. Poly(ADP-ribose) residues were marked with a PAR-specific antibody and detection followed by means of a fluorochrome-conjugated secondary antibody. The specificity of the method was proven unequivocally by a complete loss of signal when a specific PARP inhibitor (PJ34) was applied prior to irradiation-induced ribosylation. The advantage of this method is that the simultaneous determination of the DNA content in every cell allows the analysis of ribosylation events in correlation with cell cycle distribution. Based on these experiments it was found that in Imatinib-treated cells both the constitutive and the irradiation-induced poly-ribosylation are significantly enhanced. Furthermore, irradiation does not result in poly-ribosylation of all cells at a certain time: A subpopulation of cells, presumably those in the G0 resting phase, remain PAR-negative before and after irradiation. Thus, a novelty of the work at hand lies in the correlation of ribosylation events and cell cycle distribution before and after DNA damage. In this context, the central role of the Imatinib-mediated inhibition of c-Abl could also be established. The inhibited kinase activity of c-Abl seems to cause a delayed degradation of PAR. This is either caused by decreased activity of the PARP-1 antagonist PARG or by increased activity of PARP-1 itself. A disturbance of the spatially and temporally tightly modulated synthesis and degradation of PAR may lead to a prolonged interaction of PARP-1 with proteins related to SSB repair or BER, e.g. XRCC1 and DNA polymerase beta, thus resulting in the observed delay in DNA damage repair. The present study provides new insights into the impact of Imatinib on Bcr Abl-negative cells. The obtained in vitro data suggest that long-term treatment with c-Abl inhibitors may be associated with an increased likelihood of secondary neoplasias. Despite the outstanding success in Imatinib treatment of CML patients in the chronic phase, the complete elimination of the malignant clone should be the primary goal of the treatment of Bcr-Abl-positive leukemias.
Identification of essentially derived varieties in maize (Zea mays L.) using molecular markers, morphological traits, and heterosis
(2004) Heckenberger, Martin; Melchinger, Albrecht E.
The ‘breeder’s exemption’ as fixed in the UPOV convention on plant variety protec-tion allows the use of protected germplasm for the development of new plant varieties. The aim of this concept is the creation of new genetic variation to guarantee a continuous breeding progress. However, the use of molecular markers in backcrossing programs and genetic engineering has created the technical basis to develop new plant varieties without original breeding efforts. Therefore, the concept of ‘essential derivation’ was implemented into the 1991 Act of the UPOV convention to distinguish between varieties that resulted from intensive and creative selection programs and cultivars that were developed without major genetic changes from these former varieties. Accordingly, a variety is deemed to be essentially derived from an initial variety (IV), if it (i) was predominantly derived from the IV, (ii) is clearly distinguishable from the IV, and (iii) genetically conforms to the IV in the expression of it’s essential characteristics. The goal of this thesis was to evaluate and compare different approaches to assess conformity in the expression of the essential characteristics between IV and essentially derived varieties (EDVs) and to derive a theoretical and experimental basis for the devel-opment of thresholds to distinguish between independently derived varieties and EDVs in maize (Zea mays L.). The main focus was set on the evaluation of genetic distances based on ‘simple sequence repeats’ (SSRs) and ‘amplified fragment length poly¬morphisms’ (AFLPs) as well as the factors contributing to the GD between parental inbreds and their progeny lines. Furthermore, the ability of heterosis and morphological distances for identification of EDVs was examined. In detail, the objectives were to (1) analyze the factors influencing genetic distances (GD) based on SSRs and AFLPs between related maize inbred lines, (2) investigate the power of SSR- and AFLP-based GD estimates, morphological distances and heterosis for discriminating between progenies derived from F2, BC1, and BC2 populations, (3) exemplify theoretical and simulated results with experimental data, and (4) draw conclusions with regard to EDV thresholds suggested in the literature. A total of 220 flint, dent, and US maize inbred lines was genotyped with 100 SSRs equally distributed across the maize genome. The 220 lines comprised 163 triplets. A triplet consisted of one progeny and both parental lines, where the former was developed from an F2-, BC1-, or BC2 population. A subset of 58 lines (38 triplets) was genotyped addition-ally with 20 AFLP primer combinations. Furthermore, morphological traits and heterosis were observed for these 38 triplets in a field experiment over two years and three locations. The distributions of GD values for parental lines and their F2- and BC1-derived progeny overlapped for simulated as well as for experimen-tal data. Assuming that the derivation of a line from an F2 population was an accepted breed-ing procedure and the derivation from a BC1 population would not be accepted, we ob-served Type II errors (β) ranging from 0.23 to 0.37 depending on the germplasm pool for a given Type I error (α) of 0.05. For a threshold between BC1 and BC2, β ranged from 0.40 to 0.60 with an increasing tendency for higher BC levels. For fixed GD thresholds of T=0.25, 0.20, 0.15, and 0.10 suggested in the literature, substantial differences for α and β were found between different germplasm pools. Therefore, thresholds need to be gene pool specific and different thresholds for potential EDVs from intra-pool crosses than for progenies from inter-pool crosses must be applied. Discrimination of F2-, BC1-, and BC2-derived progeny lines on the basis of heterosis and morphological distances revealed β values ranging from 0.50 to 0.95 depending on the trait or combination of traits. Therefore, heterosis and morphological distances were fairly inappropriate tools for identification of EDVs due to the larger overlaps of F2-, BC1-, and BC2-distributions compared to GDs based on molecular markers. In general, SSRs and AFLPs were the most adequate tools to uncover close pedigree relationships between maize inbred lines and to discriminate among lines derived with ac-cepted or non-accepted breeding procedures. Therefore, the results presented in this study provide an example for identification of EDVs and can be transferred to other diploid crops by adjusting the corresponding thresholds.
Prediction of hybrid performance in maize using molecular markers
(2008) Schrag, Tobias; Melchinger, Albrecht E.
Maize breeders develop a large number of inbred lines in each breeding cycle, but, owing to resource constraints, evaluate only a small proportion of all possible crosses among these lines in field trials. Therefore, predicting the performance of hybrids by utilising the data available from related crosses to identify untested but promising hybrids is extremely important. The objectives of this thesis research were to develop and evaluate methods for marker-based prediction of hybrid performance (HP) in unbalanced data as typically generated in commercial maize hybrid breeding programs. For HP prediction, a promising approach uses the sum of effects across quantitative trait loci (QTL) as predictor. However, comparison of this approach with established prediction methods based on general combining ability (GCA) was lacking. In addition, prediction of specific combining ability (SCA) is also possible with this approach, but was so far not used for HP prediction. The objectives of the first study in this thesis were to identify QTL for grain yield and grain dry matter content, combine GCA with marker-based SCA estimates for HP prediction, and compare marker-based prediction with established methods. Hybrids from four Dent × Flint factorial mating experiments were evaluated in field trials and their parental inbreds were genotyped with amplified fragment length polymorphism (AFLP) markers. Efficiency for prediction of hybrids, of which both parents were testcross evaluated (Type 2), was assessed by leave-one-out cross-validation. The established GCA-based method predicted HP better than the approach exclusively based on markers. However, with greater relevance of SCA, combining GCA with marker-based SCA estimates was superior compared with HP prediction based on GCA only. Linkage disequilibrium between markers was expected to reduce the prediction efficiency due to inflated QTL effects and reduced power. Thus, in the second study, multiple linear regression (MLR) with forward selection was employed for HP prediction. In addition, adjacent markers in strong linkage disequilibrium were combined into haplotype blocks. An approach based on total effects of associated markers (TEAM) was developed for multi-allelic haplotype blocks. Genome scans to search for significant QTL involve multiple testing of many markers, which increases the rate of false-positive associations. Thus, the TEAM approach was enhanced by controlling the false discovery rate. Considerable loss of marker information can be caused by few missing observations, if the prediction method depends on complete marker data. Therefore, the TEAM approach was improved to cope with missing marker observations. Modification of the cross-validation procedure reflected, that often only a subset of parental lines is crossed with all lines from the opposite heterotic group in a factorial mating design. The prediction approaches were evaluated with the same field data as in the previous study. The results suggested that with haplotype blocks instead of original marker data, similar or higher efficiencies for HP prediction can be achieved. Marker-based HP prediction of inter-group crosses between lines, which were marker genotyped but not testcross evaluated, was not investigated hitherto. Heterosis, which considerably contributes to maize grain yield, was so far not incorporated into marker-based HP prediction. Combined analyses of field trials from multiple experiments of a breeding program provide valuable data for HP prediction. With a mixed linear model analysis of such unbalanced data from nine factorial mating experiments, best linear unbiased prediction (BLUP) values for HP, GCA, SCA, line per se performance, and heterosis of 400 hybrids were obtained in the third study. The prediction efficiency was assessed in cross-validation for prediction of hybrids, of which none (Type 0) or one (Type 1) parental inbred was testcross evaluated. An extension of the established HP prediction method based on BLUP of GCA and SCA, but not using marker data, resulted in prediction efficiency intermediate for Type 1 and very low for Type 0 hybrids. Combining line per se with marker-based heterosis estimates (TEAM-LM) mostly resulted in the highest prediction efficiencies of grain yield and grain dry matter content for both Type 0 and Type 1 hybrids. For the heterotic trait grain yield, the highest prediction efficiencies were generally obtained with marker-based TEAM approaches. In conclusion, this thesis research provided methods for the marker-based prediction of HP. The experimental results suggested that marker-based HP prediction is an efficient tool which supports the selection of superior hybrids and has great potential to accelerate commercial hybrid breeding programs in a very cost-effective manner. The significance of marker-based HP prediction is further enhanced by recent advances in production of doubled haploid lines and high-throughput technologies for rapid and inexpensive marker assays.
Toll-like Receptor 9 (TLR9) activation and the innate immune response to microbial and human DNA
(2023) Hsu, Emily; Fricke, Florian W.
The human Toll-like Receptor 9 (TLR9) is an endosomal Pattern Recognition Receptor (PRR) that recognizes DNA sequences containing the unmethylated Cytosine-Guanine (CpG) dimers, which are present in greater abundance in most bacterial genomes compared to those of vertebrates. Specific CpG-containing sequences are strongly stimulatory of human TLR9, as shown in published studies using synthetic oligonucleotides (ODN) and DNA from bacterial species of varying genomic CpG concentration. Human TLR9 activation was experimentally examined in this thesis using DNA extracted from different bacterial sources, human DNA from Caco-2 cells, known immunostimulatory ODN, and short ODN. In vitro assays using fragment length-standardized microbial genomic DNA on HEK-Dual TLR9 Cells and human peripheral blood mononuclear cells (PBMCs) revealed that TLR9 activation strongly correlated to CpG concentration of the input DNA, with an additional influence of CpG-containing 5-mer TCGTT concentration. When DNA of varying origins and fragment lengths were used together, however, complex dynamics of TLR9 activation, co-activation, and repression were observed, which were less predictable than expected from genomic CpG concentration alone. DNase I-treated microbial DNA fragments of less than 15 bp of length were non-activating on their own, but co-activated human TLR9 together with ODN-2006 in Ramos Blue (B) cells. Similarly, human DNA fragments at the length of 50-200 bp co-activated human TLR9 with both ODN-2006 and Escherichia coli DNA in HEK-dual TLR9 cells. In contrast, large human DNA fragments at over 10000 bp of length repressed TLR9 activation by ODN-2006 in Ramos Blue cells. Finally, a preliminary study was conducted in HT-29 cells on the effect of TLR9 activation on the invasion of Fusobacterium nucleatum, an opportunistic gut pathogen with a very low genomic CpG concentration at 0.296%, using ODN-2006 and human DNA as TLR9 activators. While increased presence of intracellular Fusobacterium nucleatum upon treatment with both ODN-2006 and human DNA was noted, more studies are needed to confirm TLR9 activation as a cause of greater bacterial invasion. The human colon is the location of the largest microbial population of the human body, which provides a rich source of non-human DNA in contact with human TLR9 present in intestinal epithelial cells, plasmacytoid dendritic cells (pDCs), and B lymphocytes. Additionally, the daily mass shedding and death of human intestinal epithelial cells provide large amounts of human DNA, which when combined with microbial DNA could result in co-activation and possible autoimmunity. The thesis thus provided an in vitro model of TLR9 activation by complex DNA of varying origins and fragment lengths likely to present in the human gut environment, and prepared a working basis for future studies of TLR9 activation by human fecal metagenomic DNA.