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| Identifier |
uch.biology.phd//2004boutla |
| Title |
Ανάλυση μικρών μορίων RNA που ελέγχουν μετα-μεταγραφικά φαινόμενα ρύθμισης της γονιδιακής έκφρασης |
| Alternative Title |
Analysis of short RNAS that control gene expression post - trascriptionaly |
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Author
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Μπούτλα, Αλεξάνδρα
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Thesis advisor
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Τσαγρή, Ευθυμία
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| Abstract |
During my thesis I have studied various aspects of those forms of post-transcriptional gene regulation that are mediated by various classes of small RNAs. Small RNAs are usually 21-25 nt long and interfere with the mRNA transcript in a sequence specific manner. Short interfering RNAs (siRNAs) are the result of an RNaseIII like nuclease (Dicer) on dsRNA and are functional intermediates of the posttranscriptional gene silencing (PTGS)/RNA interference (RNAi) phenomena; they direct the degradation of their target RNA. Micro RNAs (miRNA) block the translation of an mRNA through binding at its 3΄UTR. My research activities concerning these two classes of small RNA and PTGS phenomena can be divided in several subtopics. At the beginning of my PhD studies we were interested in what kind of synthetic siRNAs can induce RNAi in Drosophila embryos and how do they compare in their efficiency with long dsRNA. SiRNAs are composed of two RNA strands each having a 5΄ phosphate that form a duplex with two 3΄ protruding nucleotides at each end. Chemically synthesized siRNAs are likewise able to induce RNAi in vivo. I have tested various forms of synthetic RNA cassettes including those that are devoid of a 5΄ phosphate or they have blunt ends, which were active, although at lower efficiency than a typical siRNA. I also found that introduction of point mutations in the center of the cassettes had only moderate effect on their silencing potential. Further I studied variations in the design of the 3΄ termini of synthetic siRNAs. Secondly, I was interested to study the signal that is responsible for the systemic silencing in plants where PTGS/RNAi is non-cell autonomous. This property is also found in nematodes. I therefore fractionated RNA extracts taken from a Nicotiana benthamiana plant systemically silenced for the green fluorescent protein (GFP). These RNAs were injected into a GFP transgenic Caenorhabditis elegans line and assayed for their silencing potential. In this way I could identify an RNA component ~85 nt, in length, which could induce silencing in the nematode. Related to the plant work, my laboratory had found that potato spindle tuber viroid RNA (PSTVd) generates siRNA. This is surprising, since the viroid replication cycle takes place in the nucleus where also its dsRNA intermediates are located, while most of the PTGS components are located in the cytoplasm. In order to clarify where the siRNA accumulate in the infected host cell, we isolated nuclei from tomato plants infected with the viroid. We found that siRNAs accumulate in the cytoplasm. At current it is not clear whether they have a function in suppressing viroid replication. Finally, I became interested in miRNAs. At first we wanted to see whether it is possible to inhibit miRNA function by providing an artificial antisense target. Therefore I injected miRNAspecific antisense DNA oligonucleotides into Drosophila embryos. Some of these oligonucleotides induced specific developmental defects. In order to identify potential target genes that are regulated by those miRNAs we developed an experimental method that makes use of the DNA antisense oligonucleotides in a PCR strategy. In this way I could identify several target genes that are most likely regulated miRNA 2 and 13.
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| Language |
Greek |
| Subject |
Μετά - μεταγραφική σίγηση; RNA, Μικρά; Δροσόφιλα; RNA, Μικρά Παρεμβαλλόμενα; Γονιδιακή ρύθμιση; DNA, Ρυθμιστικά μη κωδικά |
| Issue date |
2004-12-07 |
| 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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| Views |
325 |
Ανάλυση μικρών μορίων RNA που ελέγχουν μετα-μεταγραφικά φαινόμενα ρύθμισης της γονιδιακής έκφρασης
