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And Author="Ashraf"
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| Identifier |
uch.biology.phd//2000DIS1111 |
| Title |
Transcriptional and Posttranscriptional regulation of Deoxycytidine Kinase |
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Author
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Al-Madhoon, Ashraf Said
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| Abstract |
Deoxycytidine kinase (dCK) catalyses the phosphorylation of 2'- deoxycytidine to its monophosphate forms. This is a rate liting reaction in the deoxynucleoside salvage pathway. The enzyme was found to have a broad substrate specificity: it can phosphorylate both purinr and pyrimidine deoxynucleoside substrates. dCK is also responsible for the initial activation of a number of clinically important anticancer and antiviral nucleoside derivatives. Impaired dCK expression or activity in cells usually leads to resistance to these drugs indicating that dCK plays a key role in their metabolism and pharmacological activity. For these reasons elucidation of potential regulatory mechanisms, which play a role in dCK activation, is of particular interest. In this study, we tried to understand the meshanisms that regulated dCK at the transcriptional and the posttranscriptional levels. 1. A complex pattern for the mdck regulation at the transcriptional level was detected. The mutant deletion analysis of the 5'-flanking region and the in vitro footprint exprerimentss indicated tha the proximal promoter regions might contribute to tissue specific or to cell specific expression of the gene. Our results indicated that the 5'-untranslated region might play an important role in the regulation of the gene. In transient transfection experiments this region exhibited the highest promoter activity. However, further analysis is needed to find out whether our results are compatible with the in vivo situation. 2.Although biochemical study showed tha dCK does not have isomeric forms in contrast to thymidine kinase, our study revealed the existence of a second gene with high homology to the mdck. The low levels of expression or the potential tissue specific expression gene were ostacles against cloning the cDNA of this gene. Sequence analysis indicated that this gene is intronless, which may be evolved from an intron containing progenitor gene. The insertion of such a spliced MDNA intermediate into the mouse genome could be mediated by nucleotide repeats that are detected at the 5'-upstream region. 3. Our studies on a dFdCyd resistant cell line indicate that the drug resistance phenomenon, mediated by deficiency in dCK activity, does not only involve genomic rearrangement in the dck locus, as has been described previously, but may involve posttranscriptional and poststranslational meschanisms. We also analyzed the intracellular localization of dCK by western blot assays of biochemically fractionated nuclear and cytoplasmic fractions as well as by in situ immunochemistry using highly specific C-terminal dCK was overexpressed in the cells it was mainly located in the nucleus. These results suggest the existence of a cytoplasmic retention mechanism that may involve another protein complexed with dCK. An extra project was included in this thesis that involves the improvement of the substrate specifity of the Drosophila melanogaster deoxynucleoside kinase (DmdNK). Utilizing a PCR-technique and by random sequence mutagenesis, we showed that amino acid substitutions within the Dm-dNK protein could alter its enzymatic activity toward specific substrates. The mutant kinases with the desired phenotype could be used as suicide genes therapy applications.
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| Language |
Greek |
| Issue date |
2000-10-11 |
| Collection
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School/Department--School of Sciences and Engineering--Department of Biology--Doctoral theses
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Type of Work--Doctoral theses
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| Permanent Link |
https://elocus.lib.uoc.gr//dlib/8/d/5/metadata-dlib-2000DIS1111.tkl
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| Views |
248 |
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