Browsing by Subject "Transkription"

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Functional characterization of the COOH-terminal kinase activity of the TBP-associated factor TAF1
(2006) Maile, Tobias; Sauer, Frank
Activation of eukaryotic transcription involves an orchestrated interplay between transcription factors and the general RNA polymerase II (Pol II) transcription machinery (GTM), which consists of Pol II and general transcription factors (GTFs). The GTF TFIID consists of the TATA-box binding protein (TBP) and several TBP-associated factors (TAFs). The binding of TFIID to promoters can nucleate transcription. TAF1 is the largest subunit of TFIID and plays a central role within the nucleating function of TFIID in transcription. TAF1 mediates the binding of TFIID to promoters and interacts with enhancer-bound transcription factors and several GTFs. Additionally, TAF1 contains four enzymatic activities that are essential for viability of eukaryotes and mediate posttranslational modification of GTFs and histones. TAF1 is a bipartite protein kinase and contains an NH2-terminal kinase domain (NTK) and a COOH-terminal kinase domain (CTK). A previous study demonstrated that the CTK phosphorylates serine-residue 33 in histone H2B (H2BS33). However, the role of TAF1-mediated phosphorylation in transcription regulation remained unknown. In this study, the functional importance of H2BS33 phosphorylation (H2BS33P) by TAF1 was investigated by using a combination of biochemical and in vivo assays. In vitro kinase assays uncovered the two essential kinase motifs in TAF1CTK, the ATP-binding motif and the serine/threonine-specific catalytic motif, and indicate that the TAF1 CTK has intrinsic kinase-activity. Western blot analysis using an antibody to H2BS33P revealed that H2BS33 is phosphorylated in Drosophila. RNA-interference (RNAi) assays, designed to attenuate TAF1 expression (TAF1RNAi), revealed that TAF1 is a major kinase for H2BS33 in Drosophila Schneider cells. Flow-cytometry analysis of TAF1RNAi cells indicated that loss of TAF1 expression results in cell cycle arrest in G2/M-phase. Screening the transcription of cell cycle genes in TAF1RNAi cells by using reverse-transcriptase-PCR demonstrated that the transcription of the cell cycle gene string (stg) is reduced in the absence of TAF1. Chromatin immunoprecipitation assays (XChIP) indicate that H2BS33P is detectable at the transcriptionally active stg promoter but not at the silent stg promoter in TAF1RNAi cells. These results demonstrate that phosphorylation of H2BS33 is involved in stg transcription. XChIP-assays using chromatin prepared from Drosophila embryos, which express a mutant TAF1 lacking the CTK, revealed that CTK-mediated phosphorylation of H2BS33 plays an essential role in the activation of transcription of the Drosophila segmentation gene giant. In vitro kinase assays demonstrate that Bdf1 and Bdf2, the yeast homologues of the TAF1CTK, phosphorylate histones suggesting that the kinase activity of the TAF1CTK is phylogenetically conserved. The results of this work demonstrate that TAF1CTK is a major histone kinase of H2BS33 and that TAF1-mediated phosphorylation of H2BS33 plays an essential role in the transcription events during cell cycle progression and development.
Physiological and molecular mechanisms of fruitlet abscission in mango
(2015) Hagemann, Michael Helmut; Wünsche, Jens Norbert
Compared to the typical high initial fruit set of mango (Mangifera indica L.), only a small share of those fruits reach harvest-maturity. This extensive fruitlet drop is a major yield-limiting factor, leading to substantial economic losses for mango growers world-wide. The numerous causes of fruitlet drop include infections with pests or diseases and unsuitable environmental or crop management conditions. Due to the high impact of fruitlet drop for mango production, the overall objective of this study was to further the understanding of the underlying mechanisms and to develop strategies for reducing fruitlet drop in mango. Different experimental approaches have been applied to reduce mango fruitlet drop, however, almost as numerous methods have been used for data interpretation, which makes the comparison of data between studies difficult. Therefore a model was developed for defining the timely pattern of fruitlet drop more generally, thus allowing inter-study comparisons of results. The model was tested and validated by monitoring the fruitlet drop in different management systems: traditional monocropping orchard versus 1) intercropping; 2) irrigation; and 3) plant growth regulator applications, respectively. The timely pattern of fruitlet drop was best described with a sigmoid function, which also formed the basis for defining the post-bloom, the midseason and the pre-harvest fruitlet drop stage. Results of the crop management evaluation show that intercropping of maize with mango has no detrimental effect on fruitlet drop. Irrigation resulted in approximately three times higher fruit retention compared with the non-irrigated control. A single application of 40 ppm 1 naphthaleneacetic acid at the end of the post-bloom drop stage resulted consistently in the highest fruit retention. The developed model permits for example the evaluation the treatment efficacies during midseason drop or yield forecasting at the beginning of the pre-harvest stage. It was suggested that especially during the midseason drop stage tree resources are limited, which results in inter-organ concurrence and subsequently induces fruitlet drop. This is supported by the current findings that during midseason drop mango trees show low rates of photosynthesis, which indicates drought stress. Such stress can induce ethylene-dependent fruitlet abscission. Therefore the ethylene releasing substance ethephon was used in order to study the onset and time-dependent course of fruitlet abscission. The results show that ethephon at a concentration of 7200 ppm (ET7200) is a reliable abscission inducer. The experiment was extended using ethephon at an additional concentration of 600 ppm (ET600). Both ethephon treatments reduced significantly the capacity of polar auxin transport (PAT) in the pedicel at 1 day after treatment (DAT) and thereafter compared to untreated pedicels. The transcript levels of the ethylene receptor genes MiETR1 and MiERS1 were significantly upregulated already at 1 DAT in the ET7200 while only at 2 DAT in the ET600 when compared to the control fruitlets. Specifically, a significant increase of MiETR1 in the pericarp at 2 DAT and of MiERS1 in the pedicel at 2 and 3 DAT was induced by ET600. In contrast, both genes were significantly upregulated in both tissues, except MiETR1 in the pedicel, at 1 DAT and thereafter by ET7200. The last parameter that significantly changed in response to the ethephon treatments was the concentration of sucrose in fruitlet pericarps, which was reduced at 2 DAT compared to control fruitlets. Based on these results, it is postulated that the ethephon-induced abscission process commences with a reduction of the PAT capacity in the pedicel, followed by an upregulation of ethylene receptors and finally a decrease of the sucrose concentration in the fruitlets. Ethylene receptors are key elements of abscission and other processes of the plants life cycle. Therefore the ethylene receptors were further studied at the molecular level in mango. Additionally to the previously known receptors MiETR1 and MiERS, two novel versions of the MiERS1 were identified in mango. These receptor genes, MiERS1m and MiERS1s, translate into truncated proteins with deletions of functional domains and show different expression patterns compared to MiERS1. The receptors were further studied through transient expression of fluorescent fusion proteins in the leaves of the model plant tobacco. All receptors are localized at the endoplasmic reticulum. Specific dimerization assays via bi-molecular fluorescence complementation indicate, that MiERS1m can dimerize with itself and with MiERS1, but not with MiETR1. In contrast, no dimerization of MiERS1s with the other receptors could be detected.