Doctoral theses
Current Record: 284 of 336
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| Identifier |
uch.biology.phd//1998zoumadakis |
| Title |
In vitro and in vivo studies on the action of hammerhead ribozymes |
| Alternative Title |
In vitro και vivo μελέτες της δράσης των σφυροκεφάλων ριβοενζύμων |
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Author
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Ζουμαδάκης, Μιχαήλ
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Thesis advisor
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Tabler, Martin
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| Abstract |
Ribozymes are considered those RNA molecules that have the ability to act catalytically in chemical cleavage reactions, either in cis or in trans. They can be found in several biological systems and one of the groups that are classified is that of the "hammerhead" ribozymes. One of the purposes of this thesis was to study the influence of several nucleotide deletions or changes, in the regions of the ribozyme that when base raired with those of the substrate RNA form helix I and III, on the catalytic activity of hammerhead ribozymes. For this reason catalytic antisense RNAs, whose target were regions of the Sendai virus genomic or antigenomic RNA, were constructed, bearing nucleotide deletions or mutations, resulting in imperfectly paired helices I and III. Deletions of mutations of nucleotides 2.1 or 15.1 and 15.2, abolished catalytic activity. Deletions of nucleotide 2.2 or additional nucleotides in the same region of the ribozyme, was only reducing the efficiency of the reaction. Similar results were obtained after deletion of nucleotide 15.3. Simultaneous deletions of ribozyme nucleotides in the regions forming helices I and III, resulted in cleavage of the substrate at a different site than the initially selected one. Another purpose of this thesis was the systematic investigation of the influence of the target sequence, as it is described by the NUX motif, on the cleavage rate. For this reason all the 12 different substrate combinations along with the 4 corresponding ribozymes, were constructed. All types of substrate could be cleaved, but the reaction rates as measured at 37o C, differed substantially. The best efficiency was observed with the NUC triplets, with that of auc being the best of the 12 combinatons. Finally, the in vivo action of antisense RNAs and of hammerhead catalytic antisense RNAs in the biological system of Sacharomyces cerevisiae. The target was the gcn4/lacZ or the gcn4 mRNA. The efficiency of cleavage was estimated by the b-galactosidase enzyme assay or the sensitivity of the cells to 3-amino-triazol (AT). Only in one case, where the gcn4/lacZ mRNA was expresssed from a plasmid, a 50% reduction in the enzymatic activity was observed, which was not accompanied by a reduction of the steady-state mRNA levels.
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| Language |
Greek |
| Subject |
RNA ΩΣ ΒΙΟΛΟΓΙΚΟΣ ΚΑΤΑΛΥΤΗΣ; ΑΝΤΙΚΩΔΙΚΟ RNA; ΚΑΤΑΛΥΤΙΚΑ RNA; ΣΦΥΡΟΚΕΦΑΛΟ ΡΙΒΟΕΝΖΥΜΟ |
| Issue date |
1998-04-02 |
| 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/9/4/1/metadata-dlib-1998zoumadakis.tkl
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| Views |
378 |
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