Altklausur Flashcards
Explain miRNAi.
last edit: Inga, 8.3.
- RNAi = interference RNA, is used for gene silencing
- RNAi either blocks/inhibits translation or destroys mRNA
- > this mechanism is part of a natural response of the host that most likely evolved to control viruses or transposon replication
- miRNAi/microRNA are single-stranded, genomically encoded non-coding RNAs
- processed by enzyme Dicer, interact with RISC-complex
- mature miRNAs are similar to short interfering RNAs (siRNAs) produced from exogenous double-stranded RNA (dsRNA)
Explain Gal4/UAS.
last edit: Inga, 8.3.
- system commonly used in Drosophila to drive expression of a gene of interest
- GAL4 protein (derived from yeast) is expressed in a cell- or tissue-specific pattern and is kept with an endogenous promoter/enhancer in one fly line, the so-called driver (GAL4 expressing) line
- Upstream Activation Sequence (UAS) is an enhancer that is specific to GAL4 and is kept together with a specific gene of interest in another fly line, the responder (UAS-target gene) line -> gene of interest is silent in the absence of GAL4!
- when these lines are crossed, in the progeny of this cross GAL4 protein will bind to UAS and drive the expression of the target gene in a cell- or tissue-specific pattern
- other GAL4 systems:
> TARGET system: uses GAL80 for temporal control of GAL4-UAS-based
> inducible GAL4 system: GAL4 is coupled with a hormone receptor -> transcriptionally active in the presence of the hormone, transcriptionally inactive in the absence of the hormone
> split-GAL4 system: GAL4 is split up -> transcriptionally active when expressed in the same cell, otherwise silent
Which line was used as experiment and control group in confocal?
(last edit: Inga, 8.3.)
For immunofluorescence staining:
- brain:
> Orco-Gal4 (III) x UAS: mcD8-GFP (III)
> GH146-Gal4 (III) x UAS: mcD8-GFP (III)
> MB247-Gal4 (III) x UAS: mcD8-GFP (III)
-> aim of experiment: localization of Drep2 & BRP at the olfactory pathway synapses; visualization of Orco-Gal4, GH146-Gal4 & MB247-Gal4 expression patterns (olfactory pathway)
- NMJ:
> Ok6-Gal4, UAS:dicer2 x UAS:BRP-RNAi (B3/C8) (III)
> Ok6-Gal4, UAS:dicer2 x W1118 (PFA fixation)
> Cac::sfGFP/X
-> aim of experiment (Ok6-Gal4 lines): localization of BRP & GluIID, verification of BRP-RNAi and function of BRP regarding synapse assembly & function
-> aim of experiment (Cac): localization of Ca2+ channels within synapse & visualizing the resolved architecture of BRP ring around Ca2+ channel
- ERG:
> GMR-Gal4(X) x UAS-BRP-RNAi (B3/C8) (III)
> GMR-Gal4(X) x W1118
> could NOT be used: GMR-Gal4(X) x UAS:ButaliniumToxin (III) because effects of toxine are lethal
-> aim of experiment: determination of BRP downregulation phenotype regarding synapse function of eye synapses (+ effect of blocking NT release using Butalinium toxine in eye synapse)
How to read ERG?
last edit: Inga, 8.3.
- wild-type flies show stereotypical ERG response:
> light stimulus elicits (hervorrufen) upward voltage spike (= on-transient) from laminar neurons
-> depolarization (curve goes down) of photoneurons elicited from photoreceptor cell (= receptor potential)
-> after end of light stimulus downward spike (= off-transient, repolarization off laminar neurons)
-> voltage rapidly returns to baseline - in BRP knockdown: BRP is a presynaptic protein that is essential for formation of AZ & therefore signal transmission
> signal from photoneurons won’t reach laminar neurons
-> in curve of ERG there won’t be an on-transient or off-transient
Explain the three steps in PCR.
PCR stands for “polymerase chain reaction” and it’s a method used for DNA amplification. The procedure consists of three major steps.
1. Denaturation: The DNA that is supposed to be amplified gets denaturated at a high temperature (~95°C), so the primer can later on bind to the single strand). The hydrogen bonds between the complementary bases are broken and the double-stranded DNA template gets split up into two single-stranded DNA molecules.
2. Annealing: The temperature is reduced to so-called “annealing temperature” (usually ~60°C) of the specifically designed forward and reverse primer. The temperature has to be low enough to allow hybridization of the primers to the DNA stand and high enough for the hybridization to be specific.
3. Elongation: Temperature depending on DNA polymerase that is used in the setup. Polymerase synthesizes a new DNA strand complementary to DNA template strand by adding deoxynucleoside triphosphates (dNTPs) from the master mix that are complementary to the DNA template in 5’-to-3’ direction.
These three steps are one cycle. For proper amplification, several cycles are necessary.
Explain FRAP technique.
FRAP stands for Fluorescence Recovery After Photobleaching and is a technique to measure molecular diffusion and active processes in time. Using FRAP can help to understand to which extent trafficking vesicles can move between different compartments of cells, to see the speed of response and the amount of cargo carried. Moreover it enables to gain insight into binding and release kinetics of molecules with particular substrates or surfaces to bind.
For FRAP, the region of interest (ROI) is bleached with a laser, removing all fluorescence from this area but not removing the molecules themselves. After time, the fluorescence comes back to the ROI by new molecules diffusing into it. The diffusion coefficient can be determined by measuring the course of fluorescence intensity over time. The later the original fluorescence of the ROI is reached again, the slower is the diffusion of the fluorescing molecule(s).
Molecular kinetics can be fast or slow processes (measured in X = time, Y = distance) and they are visualized in a so-called kymograph.
Explain CRISPR.
- Clustered Regularly Interspaced Short Palindromic Repeats
- bacterial mechanism to defend against viruses, used for genome editing
- allows deletion, insertion and/or replacement of bases at a specific gene locus (and controls gene expression)
- enables multiplexing: several mutations in the same cell at the same time
- essential components: CRISPR-RNA (crRNA) resp. guideRNA (gRNA), Cas9 enzyme and PAM (Protospacer Adjacent Motif) sequence
- > design of simple chimeric gRNA/cRNA to target any sequence next to a PAM (NGG, NCC) in the genome
- > gRNA binds to Cas9 enzyme and this complex binds to target sequence
- > Cas9 cuts DNA by creating a double-strand break (DSB) in the genome, 3bp upstream of PAM
- > DSB is afterwards repaired by cell by either non-homologous end joining (NHJEC) or homolohy-directed repair (HR)
- > control via PCR
description of the procedure in Fragenkatalog:
- > search for homologous sequence for designed gRNAs (many Gs and Cs) to bind to
- > gRNAs detect homologues areas via PAM structure
- > homologous arm are designed
- > Cas9 endonuclease cuts genomic target DNA and creates DBS
- > DBS can be repaired by homologous recombination with cassette that is to be inserted or by non-homologous recombination
- > single strand breaks are repaired by homologous recombination with the integrated cassette
- > control via PCR
Name advantages and disadvantages of drosophila as experiment animal.
ADVANTAGES:
- small genome, only four chromosomes
- all stages genetically investigated > 100 years
- genome known (fully sequenced)
- low maintenance cost
- fast reproduction cycle/short life cycle
- smaller space requirements
- genome is relatively easy to manipulate
DISADVANTAGES:
- research results not (100%) transferable to humans as it’s not a mammalian system
- high risk of contamination through other genotypes
- material can’t be frozen easily for later use (like bacteria)
- handling requires more training due to small size
Draw olfactroy input/ouput?
- odor binds to olfactory receptors (OR) in dendrites of olfactory receptor neurons (ORN) in antennae and maxillary palps
- > transported as AP via axons of ORN to antennal lobes where there are ~50 glomeruli with synapses
- > antennal lobes are surrounded by soma of the olfactory projections neurons ([O]PNs), dendrites of the PNs are in the glomeruli
- > each PN generally receives information from a single glomerulus
- > axons of PNs send information to higher brain centers, the mushroom body (MB, center of learning and memory) and the lateral horn (responsible for innate responses)
- > in the MB: Calyx (= dendrites of the Kenyon cells that are the soma of the MB neurons) receives information from PNs (+ MB lobes are axons of Kenyon cells, there are α/β, α’/β’ and γ -> α and β are originated from the same cell; α and α’ are vertical; β, β’ and γ are horizontal; γ do not branch!
- > processing of information and behavioural output (e.g. approach or avoidance) OR innate response (lateral horn)
How can STED microsocopy have better quality/resolution?
STED stands for Stimulated Emission Depletion. A STED microscope is a fluorescence microscope that is equipped with two lasers, an excitation laser and a depletion laser (STED laser).
The resolution increase is obtained by shrinking the point spread function (PSF) of the microscope by depleting the fluorescence emission in the periphery of the diffraction limited spot using the phenomenon of stimulated emission depletion. The emission in the sample can only occur at the center of the PSF which is obtained by using a donut-shaped STED laser (shape is achieved with PhaseMod) with a wavelength longer than the emitted fluorescence. This allows a resolution given by the spot size of the remaining excited fluorophores. Resolutions of 30-50nm have been achieved with organic fluorophores.
What’s the difference/similiarty between the mouse AZs and drosophila AZs?
DIFFERENCES:
- level(s) of genetic redundancy -> knockout of one component has much worse effect in Drosophila
- AZ of mice is more complex
SIMILARITIES:
- similar components -> either the same (e.g. RIM-BP) or homologues (BRP in flies, CAST/ERCs in mice)
