Additional Flashcards
Transformation
One helper protein coding for transposase without a recognition sequence (to prevent insertion) and a P-plasmid containing transposase recognition sequences, a marker gene, the gene of interest and an e-coli selectable marker gene are injected into an early stage embryo -> random transposition
Transposition needs to be on the germ line to be passed on -> marker gene in next generation gives evidence
P-Element Mutagenesis
Random transposition can lead to a loss of a gene at the insertion site
–> Insertion tends to occur near actively transcribed genes as the chromatin structure is loosest here
Enhancer Trap
A recombinant transposon construct that is inserted into the genome and will express a reporter gene product only in certain cells when its inserted near an enhancer that is specific for these cells.
Designing a Primer
- 18 to 25 nucleotides or more
- End with double G/C (GG, GC, CC, CG)
- melting temperature of forward and reverse primer should not be more than 5°C apart
Primer in 5’-3’ direction, complementary strands
Where do different Driver lines express in the olfactory system?
Orco-Gal4 (III) x UAS: mcD8-GFP (III)
Orco>GFP
Olfactory receptor neurons
GH146-Gal4 (III) x UAS: mcD8-GFP (III)
GH146>GFP
Olfactory projection neurons
MB247-Gal4 (III) x UAS: mcD8-GFP (III)
MB247>GFP
Mushroom body neurons
How can you verify by PCR (or phenotypic markers) which fly contains a desired DNA sequence?
Phenotypic markers: e.g. GFP
PCR: Design Primers that are within the desired DNA sequence or flanking it –> PCR won’t work if the DNA sequence where the primer binds isn’t present
What is the normal resting membrane potential in mV of muscle 6 at the Drosophila NMJ during electrophysical recordings?
-60 to -70mV
What is an mEJC or miniature excitatory junctional current?
An mEJC is the postsynaptic response to the SPONTANEOUS (i.e. no stimulus) presynaptic release of a single synaptic vesicle
When we do a paired pulse experiment, what are we referring to and what can we determine from this type of experiment?
A paired pulse experiment is an experiment when two consecutive stimulations are given, with a short inter stimulus interval (in our case 10ms or 30ms). This type of experiment is used to gain some insight into short-term plasticity and vesicular release probability.
Briefly describe the function of the three electrodes that are used in Two Electrode Voltage Clamp (TEVC) electrophysical recordings.
Two sharp microelectrodes are used during normal recordings, one sharp microelectrode is used to constantly record the membrane potential of the cell, whereas thesecond is used to pass current into the cell to ‘clamp’ the membrane potential at the predetermined command potential. A third fire-polished suction electrode is used to stimulate the motor axon innervating the muscle being recorded.
What is the most important cation for proper synaptic transmission at the Drosophila NMJ? This cation is also added to the bath solution to mediate transmission. What is the approximate physiological extracellular concentration of this cation?
CA2+ is the most important cation for synaptic transmission at the Drosophila NMJ. During TEVC recordings, CaCl2 is added to the bath solution at the approximate physiological extracellular concentration of 1-1.5 mM
How would one determine the approximate number of synaptic vesicles released following a single action potential?
One would first have to determine the amplitude of the postsynaptic response to the release of a single synaptic vesicle. This can be accomplished via mEJC recordings. Once the amplitude of a single vesicle release event is determined, one can then use a stimulation protocol to determine the evoked excitatory junctional current (eEJC) amplitude, which is the postsynaptic response to presynaptic release of MANY synaptic vesicles following initiation of an action potential. The eEJC amplitude can then be divided by the average mEJC amplitude to determine the approximate number of synaptic vesicles that were released following a single action potential.
RISC Complex
RNA-induced silencing complex
-active part: argonaute proteins –> cleave the target mRNA strand complementary to their bound siRNA
Dicer
specialized ribonuclease that processes dsRNA into small RNA fragments
-composed of three structurally rigid regions connected by flexible hinges and propose that conformational flexibility facilitates dsRNA binding and processing
- ATP-dependent RNA helicase domain
- RNAse III motifs
- dsRNA binding domain
Co Immunoprecipitation
Proteins are isolated from a mixture using antibodies coupled with beads. The antibody binds the antigene (protein). After washing and elution only the targeted protein and its interaction partners are left and can be analyzed.
