Toolbox/Lecture 5

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WIKIPEDIA BIO154/254: Molecular and Cellular Neurobiology

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Contents


Lecture 5

Enhancer Promoter Screen

A screen for over expression mutant phenotypes. The genotype is created through random insertion of a strong promoter into the genome.

Lethal Enhancer Screen

A screen for a second mutation that enhances a phenotype of another mutation which by itself is not lethal. In other words, to screen for a second mutation that by itself is viable but when combined with the first mutation produces a lethal phenotype. This type of genetic screen is usually used to delineate gene interactions because the second mutation must be involved in some pathway with the first in order to it be a lethal enhancer. Such a genetic screen usually use a chemical mutagen (such as EMS) to induce the second mutation and then crossing the mutated flies with those with the first mutation. Those with desired lethal phenotypes were then selected.

In lecture 4, ubiquitination processes were implicated in the development of synapses at the Drosophila neuromuscular junction. DiAntonio et al (Nature, 2001) showed that overexpression of fat facets, a deubiquinating protease, led to disrupted synaptic function and an overgrowth of synapses. A lethal enhancer screen was employed to find other genes that enhance the fat facets phenotype. The screen looked for viable mutations that produced lethal phenotypes when combined with fat facets overexpression. The Highwire gene was one such mutation isolated in the screen. Highwire loss of function mutations resulted in the same synaptic overgrowth as fat facets overexpression and biochemical analysis of highwire revealed it to be an ubiquitin ligase. Take together, these data show that fat facets and highwire antagonize each other in the ubiquitination process and strongly support the idea that synaptic development is controlled by positive (highwire) and negative (fat facets) regulators of ubiquitination.

Lecture 5 Model Systems

Electric eel

The electric eel (Electrophorus electricus) is a species of fish native to South America that is able to generate electric shocks using the electric organ of its body. The organ is composed of electroplaques, a stack of plates that can generate charges. The organ is also extremely rich in voltage-gated sodium channels. In 1986, a team of Japanese researchers led by Shosaku Numa cloned the cDNA for the sodium channel using the electric organ of the eel. From that scientists were able to deduce the biochemical structure of the sodium channel.

Lecture 5 Techniques

Aldicarb

Aldicarb (chemical name: 2-methyl-2-(methylthio)propionaldehyde O-methylcarbamoyloxime) is an carbamate-class insecticide applied directly to the soil and is used to control mites, nematodes, and aphids. In the laboratory, aldicarb acts as an inhibitor of acetylcholinesterase (AChE), the enzyme present in the basal lamina of the post-synaptic cell of the neuromuscular junction (NMJ) which breaks down the neurotransmitter acetylcholine (ACh). ACh is the excitatory neurotransmitter for muscular contraction; therefore, inhibition of AChE using aldicarb results in prolonged activation of ACh receptors in the post-synaptic cell, causing paralysis and eventually death.

Aldicarb is a useful tool in genetic screens that search for mutants resistant or hypersensitive to the aldicarb-induced paralysis. These mutants will likely have mutated genes involved in the ACh signaling pathway at the NMJ. Mutants resistant to aldicarb will likely have an impairment of normal ACh signaling, while mutants hypersensitive to aldicarb will likely have an exaggeration of normal ACh signaling.

PCR

PCR, or the polymerase chain reaction, is an experimental technique devised by Kary Mullis in 1984 and is used to amplify a targeted DNA segment for further experimental analysis. PCR does not require knowledge of the target DNA sequence; however, knowledge of the DNA sequences flanking the target is necessary.

A PCR cycle proceeds as follows: 1) The parent DNA duplex is separated by heating the solution to 95°C for 15 seconds, exposing the bases on each strand. 2) Primers for the flanking DNA sequences anneal to the 3'-end of each parent DNA strand when the solution is cooled to 54°C. 3) A heat-stable DNA polymerase called Taq DNA polymerase (derived from the thermophilic bacterium Thermus aquaticus) synthesizes complementary DNA strands starting at the primers and using available nucleotides in solution when the solution is heated to 72°C. Repeated cycles of PCR allow for an exponential amplification of the target DNA sequence.

E. coli

Ion replacement

Ion replacement experiments involve changing the composition of a solution such that a certain ion is replaced by an equimolar amount of a similar ion. For example, an NaCl solution could be replaced with a LiCl solution, a RbCl solution, a NH4Cl solution, etc. These experiments allow one to determine the specificity toward a certain ion, such as the specificity of membrane-based sodium pores towards sodium. This technique was used to measure the role of sodium ions in triggering action potentials in squid neurons (Slide 23).

Low transmitter release conditions

Low transmitter release conditions (low calcium, high magnesium) were used to show that the distribution of endplate potentials matches a Poisson Distribution (Nicholls et al.).

Caged calcium

Caged compounds are biologically inactive compounds that can be loaded into cells and then released in their active form. This "uncaging" is accomplished by a flash of light (approximately 360nm). Absorption of the photons' energy causes the inactive, or "caged" compound to undergo a reaction to create the active compound. These reactions typically take place within a few milliseconds. Caged compounds are extremely useful in studying important sigalling events, such as the release of neurotransmitters, and activation of membrane receptors. Calcium plays a significant role in regulating synaptic activity. Therefore, using caged calcium is quite effective in studying neuronal activity and signalling.

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