The Mechanisms Linking Ribosomal Translation And Pre Mrna Processing

10128 Words Jul 28th, 2015 41 Pages
Investigation of the mechanisms linking ribosomal translation and pre-mRNA processing in yeast

Nine-Month Report submitted by
Marija Petrić
ID: 1454106

Supervisor:
Dr Saverio Brogna
Contents

Contents
1. Introduction 3
1.1. Eukaryotic gene expression 3
1.2. Quality control of eukaryotic gene expression 5
1.3. Nonsense mediated mRNA decay 6
1.4. Factors involved in NMD 7
1.5. NMD models 17
1.6. Ribosome release model 21
1.7. Role of UPF1 in the nucleus 23
2. Objectives 25
3. Materials and methods 27
3.1. Schizosaccharomyces pombe strains 27
3.2. S. pombe media 27
3.3. S. pombe culturing and maintenance 27
3.4. PCR based gene targeting – one step PCR approach 28
3.5. PCR based gene targeting – two step PCR approach 30
3.5. Agarose gel electrophoresis and gel extraction 32
3.6. Transformation of S. pombe strains for PCR based gene targeting 32
3.7. Colony PCR 33
3.8. pDUAL expression vector 34
3.9. Polysome profile 36
3.9.1. Preparation of sucrose gradients 36
3.9.2. Cell lysis 36
3.9.3. Sedimentation of translation complexes 37
3.9.4. RNA and protein precipitation from polysomal fractions 38
3.10. SDS-PAGE and Western blot 39
4. Results 41
4.1. How does UPF1 bind its substrates? 41
4.1.1. Introduction 41
4.1.2. Results and discussion 43
4.2. What is the mechanism of NMD? 48
4.2.1. Optimisation of polysome profile analysis 48
4.2.2. Ribosome release model 49

1. Introduction
1.1. Eukaryotic gene expression Gene expression is the basic cellular process which converts…
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