Describe the life-cycle of Drosophila melanogaster (developmental stages and times)!
adult -> fertilized egg -(cleavage)-> syncytial blastoderm -(gastrulation)-> embryo ->(hatching)-> 1st instar larva (ca. 1 day after fertilization) -> 2nd instar larva (ca. 2 days af) -> 3rd instar larva (ca. 6 days af) -> pre-pupa (for ~2 days) -> pupa (for ~3 days) -(metamorphosis)-> adult
- at 25°: 10-12days; slower at <25°; faster at >25°
What is a balancer and what is its function?
= balancer chromosome
- chromosomal rearrangements that allow lethal mutations to stably maintain in heterozygotes
- > if an allele is lethal as homozygotes, balancers are used to keep the stock viable
- > balancers are lethal as homozygotes and in recombination
- inhibits crossing-over
- for tracking: marker on balancer chromosome (can be identified phenotypically or are lethal as homozygotes)
Name and describe three adult markers of Drosophila!
last edit: Inga, 6.3.
- colour: wildtype, ebony -/- (e), yellow -/- (y)
- eyes: white -/- (w), mini white (w+), Bar +/- (B), Drop +/- (dr), Irregular Facets +/- (If)
- wings: Curly +/- (Cy), Serrate +/- (Ser), vestigial -/- (vg)
- bristles: Humeral +/- (Hu), Pin+/- (Pin), Stubble +/- (sb), singed -/- (sn)
Describe 3 advantages and 3 disadvantages of Drosophila as a model system!
ADVANTAGES:
- small genome
- known genome
- low maintenance costs
- saves space
- relatively easy to manipulate
- short life cycle
DISADVANTAGES:
- research results not (100%) transferable to humans because its not a mammalian system
- high risk of contamination through other genotypes
- material can’t be easily frozen like bacteria
- handling requires more training due to small size
What is a P-element?
= transposable P-element
- transposable elements are so-called „jumping genes“ that can move from one position in the genome to another
- transposable P-element is one of the best studied mobile DNA elements in eukaryots (eukaryotic cut-and-paste-transmission)
- needs enzyme Transposase
- length of 2907 bp with four exons and three introns
- 31bp terminal inverted repeats (TIR)
- 11bp internal inverted repeats (IIR) located ~100bp from the ends
What are micro-RNAi and which function do they have?
- RNAi = interference RNA
- RNAi is used for gene silencing either blocks/inhibits translation or destroys mRNA -> mechanism is part of a natural response of the host that most likely evolved to control viruses or transposon replication
- microRNAs are single-stranded, genomically encoded non-coding RNAs
- processed by enzyme Dicer, interact with RISC-complex
- mature miRNA are structurally similar to siRnA produced from exogenous dsRNA
Name and explain the different memory phases exist in Drosophila!
- short-term memory: for a few minutes, does not require repetitive training, in experiment flies are tested 5 min after exposure to stimuli
- mid-term memory: for up to few hours; in experiment flies are tested 1h after exposure to stimuli or 3h if interested in ARM and ASM
- anesthesia resistent/sensitive memory: 2,5h after exposure to stimuli, flies receive a 90s cold shock followed by 30 min recovery time
- > ARM: ability to keep memory after shock
- > ASM= MTM – ARM
- long-term memory: for 24h up to a few days, requires repetitive training (e.g. 10 times training session); in experiments flies are tested 24h after training
Describe appetitive learning and aversive learning!
- appetitive learning: positive unconditioned stimulus (US) paired with conditioned stimulus (CS+) vs. unpaired conditioned stimulus (CS-)
- > e.g.: US = food/sugar, CS+ = odor A, CS- = odor B; training; test = choice between A and B; A should be chosen
- aversive learning: negative unconditioned stimulus (US) paired with conditioned stimulus (CS+) vs. unpaired conditioned stimulus (CS-)
- > e.g.: US = salt/bitter taste/electric shock, CS+ = odor A, CS- = odor B; training; test = choice between A and B; B should be chosen
What is neuropil and which neuropil in insects is known as a center for learning and memory? Describe the structure.
- neuropil: region/structure of high synaptic densitiy with relatively low amount of cell bodies (soma) -> between glia
- in insects: mushroom body (MB)
- MB is innervated by olfactory projection neurons and the MB neurons consist of:
- > Kenyon cells = clustered, ~2.500 in each hemisphere, are the soma of the MB neurons
- > Calyx = claw-like, are the dendrites of the Kenyon cells (receive information from axons)
- > MB lobes = α and α’ (vertical), β and β’ (horizontal), γ (horizontal, do NOT branch/bifurcate) -> α and β are originated from same cell
Name three proteins implied in building a synapse. What are their functions?
- BRP: cytoskeletal protein, e.g. important for integrity of T-bars at pre-active zones, contributes to Ca2+ channel clustering
- Unc13 (release factors Unc13 A/B): essential for synaptic transmission, different isoforms can be found in different concentrations in different synapses
- RIM-BP: regulation of Ca2+ channel accumulation (“fine tuning”)
- SNAP-25: component of trans-SNARE complex -> important for specificity of membrane fusion, executes fusion by bring SV and plasma membrane together
- SNARE proteins: mediate vesicle fusion
- Neurexin-1 (NRX-1): required for synpase formation
- Synapse defective-1 (Syd-1): together with Liprin-α “master organizer” of synaptic differentiation
Describe the olfactory system in adult flies.
odor binds to olfactory receptors (OR) in dendrites of olfactory receptor neurons (ORN) in antennae and maxillary palps
->
transported as action potentials via axons of ORNs to antennal lobes where there are ~50 glomeruli with synapses
->
antennal lobes are surrounded by soma of olfactory projection neurons (OPN)
->
axons of OPN send information to mushroom body (MB, learning and memory) and lateral horn (innate response)
->
in the MB: Calyx (=dendrites of the Kenyon cells that are the soma of the mushroom body neurons) receives information from OPN and process // OR: lateral horn -> innate response
=> behavioural output
What is a bouton?
last edit: Inga, 6.3.
A (small) swelling at the terminal ends of axons containing an AZ with synaptic vesicles and neurotransmitters.
What is an Active Zone? What is the active zone scaffold and what is its function? Name three major components.
(last edit: Inga, 6.3.)
The active zone AZ is the region of the presynapse where SV fuse with the presynaptic membrane and neurotransmitter release is initiated. The AZ scaffold describes several proteins that stabilize, support and ease vesicle fusion, Ca2+ clustering etc.
-> BRP, RIM, RIM-BP, Syd-1, Unc13A, Cacophony…
What is the difference between confocal microscopy and widefield micrscopy?
(last edit: Inga, 6.3.)
- confocal microscopy:
> higher in-depth resolution with less background and better contrast
> several sample z planes are scanned (point to point -> image), images can be stacked to high resolution 3D image that can be digitally processed
> confocal microscope has pinhole: essential for 3D - widefield microscopy
> entire sample is illuminated (no z planes) -> more noise because background is illuminated, too
> only 2D image
What is CRISPR technique? Explain the system and the application.
- 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)
- essential components: CRISPR-RNA (crRNA), Cas9 enzyme, PAM sequence
- > design single chimeric guide RNA crRNA/gRNA to target any sequence next to a PAM (Protospacer, Adjacent Motif, NGG/NCC) in the genome
- > gRNA also binds to Cas9
- > Cas9 cuts DNA by creating a double-strand break in the genome -> occurs on both strands, 3bp upstream of PAM
- > DSB is repaired by either non-homologous end joining (NHEJ) or homology-directed repair (HR)
- > control via PCR
ADDITIONAL:
- desired sequence can be incorporated into target DNA with marker genes (e.g., dsRed) (by homologous arms)
- by crossing and screening for dsRed it is examined with the help of PCR whether CRISPR worked (with the help of the homologous arms)
What is axonal transport and what is its function?
last edit: Inga, 6.3.
The movement of synaptic vesicles, proteins, lipids and mitochondria through the cytoplasm of the axon. It enables the transport neuronal proteins between the soma and the synapse. The transport from the soma to the synapse is called “anterograde” and from the synapse to the soma “retrograde”. We distinguish two types:
Slow axonal transport that carries enzymes etc. that are not quickly consumed and utilizes the axoplasmic flow. And fast axonal transport that utilizes kinesins, dyneins and microtubules and actively “walks” vesicles up or down an axon.
Is it possible to simultaneously image two proteins in vivo? How would you label them? Explain in this context the principle of fluorescence.
(last edit: Inga, 6.3.)
Yes, the proteins can be labeled with different fluorophores that have different emission (and excitation) spectra (e.g. GFP and RFP).
Flurorescence is the ability of a fluorophore to emit light after getting excited by light of a certain wavelength - hereby the emitted light always has a longer wavelength as it loses vibrational energy during the state of relaxation after the excitation.
How do you determine experimentally the differences in spatial coupling of synaptic vesicles to voltage gated calcium channels? (Mouse)
(Ergänzung nötig! last edit: Inga, 6.3.)
By the use of the (slow) Ca2+ chelator EGTA (and BABDA). When an AP reaches the presynapse and opens the voltage-gated Ca2+ channels, EGTA binds the inflowing Ca2+. Ca2+ will not reach the SV in the periphery and therefore, these vesicle will not fuse with the presynaptic membrane and the enclosed NTs can’t be released into the synaptic cleft.
Rebecca: This leads to a decreased amplitude of the eEJC.
-> IF staining for Ca2+ channel + release factor -> STED (Kommentar von Thorsten auf meinem Test…)
When we do a paired pulse experiment, what are we referring to and what can we determine from this type of experiment?
(last edit: Inga, 6.3.)
In a paired pulse experiment, two consecutive stimuli are given with a short interstimulus interval (ISI, 10-30 ms) and the electrical response in the postsynapse is recorded.
It is used to investigate synaptic short-term plasticity and vesicular release probability.
What is the neurotransmitter at the Drosophila NMJ? What are other neurotransmitters?
(last edit: Rebecca, 11.3.)
- glutamate at Drosophila NMJ (excitatory)
- other: dopamine, serotonine (are neuroMODULATORS, excitatory and inhibitory); GABA (inhibitory, generelly at matured synapse)
-Acetylcholin (in ZNS, excitatory)
What is a PCR and what is it used for?
last edit: Inga, 6.3.
- polymerase chain reaction
- used for DNA amplification
- components include specifically designed primers (forward & reverse)
- 3 major steps:
1. denaturation: DNA is denaturated at high temp. to enable primer binding
2. annealing/hybridization: temp. is reduced to primer annealing temp. (usually ~60°C), primers can bind to DNA
3. elongation: temp. depends on DNA polymerase used, polymerase synthesizes new complementary DNA strand -> DNA gets amplified - results are afterwards investigated in gel-electrophoresis
