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Advanced Biochemistry. Übungsfragen

Prüfungsvorbereitung 2014 12 Seiten

Chemie - Biochemie

Leseprobe

1. Wahl

a) Group II introns and eukaryotic nuclear pre mRNA splice via the same reaction mechanism. Why do eukaryotic introns need spliceosome while group II intron can self-splice?

b) What could be advantages of using the spliceosome?

a) group II introns have a very strict tertiary structure that brings splice sites into the correct position. Eukaryotic splice sites are degenerate and weak. They have to be recognized by multiple factors of the spliceosome.

Group II introns are highly conserved in sequence and

structure – no flexibility for splice site choice

Nuclear introns harbor only a few short (and often degenerate) signal elements – high flexibility for splice site choice

b) This concept provides a flexibility in splice site choice and enables alternative splicing. Plus, stepwise assembly provides many potential checkpoints.

2. Wahl

You found a splicing inhibitor “SplicEx” (dissolved in 20% DMSO).

a) Describe assay to investigate which step of the splicing is inhibited by SplicEx (incl. control, important reagents, analysis,...)

b) What would you see in your assay if SpliceEx stalled splicing before the catalytic active complex is formed?

a) conduct in vitro splicing experiment with MBP-MS2 tagged pre-mRNA and either SplicEx (experiment) or (DMSO) control (plus everything that is needed: spliceosome, ATP,…)

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RNA analysis of affinity-purified stalled splicing complexes by denaturing gel electrophoresis and silver staining

b) complex B: U1, U2, U4, U5, U6, unspliced pre-mRNA

Vgl. BA3 oder SAHA Vorlesung (Ich hab SAHA beschrieben, was korrekt ist…)

3. Heyd

a) Describe two methods for genome-wide analysis of alternative splicing.

b) What has to be done afterwards? Describe one methode in detail

a) exon array on splicing sensitive microchips that contain probes and can be detected by fluorescence when bound to target

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RNA Seq/Deep Sequencing where RNA is reverse transcribed into cDNA, DNA shearing, adapter ligation, PCR, quantify PCR products according to their sequence

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b) Validation

RT-PCR and either qPCR with 32P-forward primer in one tube and gel electrophoresis or isoformspecific primers in separate tubes

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4. Freund

a) An antigen is presented on a APC. What happens?

b) What enzymes and catalysts are needed to present antigen-peptides?

a) T cells (CD8) are activated, proliferate and look for other cell that present this antigene to kill them

b) HLA-DM catalyses exchange of CLIP (Platzhalter) and antigen peptide by stabilizing open conformation of MHC II

Heinemann

a) What is a molecular machine?

b) What is an AAA ATPase? Give an example.

c) How is p97 different from other AAA ATPases?

a) A molecular machine is any discrete number of molecular components that produce quasi-mechanical movements (output) in response to specific stimuli (input).

b)

- ATPases associated with a variety of cellular activities
- Hexameric molecular machines
- AAA proteins couple chemical energy provided by ATP hydrolysis to conformational changes which are transduced into mechanical force exerted on a macromolecular substrate.
- Examples: p97, dynesin

c) p97 is a hexameric ATPase of the AAA family which disassembles SNARE proteins after membrane fusion

Daumke

a) What is the function of ras and dynamin GTPases? (keyword)
b) Compare the families in respect to their affinity and activity.
c) Explain experiments showing these differences (what parameter is measured, set up)
d) Why are they different (functional use)?

a) GTP hydrolysis
b) comparison:

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c) Dynamin oligomerizes around lipids: Provide liposome (experiment) or no lipids (control) and GTP to dynamin and measure inorganic phosphate release (colorimetric assay → change in absorbence can be measured with spectrometer)

d) ras has to be activated for quite a time to be able to activate other proteins. Dynamin releases vesicles, that has to happen fast to refill SV pool quickly.

[...]

Details

Seiten
12
Jahr
2014
ISBN (eBook)
9783656823490
Dateigröße
1.4 MB
Sprache
Deutsch
Katalognummer
v282482
Institution / Hochschule
Freie Universität Berlin – Institut für Chemie und Biochemie
Note
1,0
Schlagworte
advanced biochemistry übungsfragen
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Titel: Advanced Biochemistry. Übungsfragen