Genetics Problem Spaces

Introduction Problem Spaces Behavioral Genetics Overview Intro Exercise Research Questions Literature Resources Assignments Student Projects

Behavioral Genetics Introductory Exercise Introduction The nematode Caenorhabditis has been successfully used to dissect the genetic basis of an array of complex biological phenomena. Recently a Nobel Prize in Medicine was awarded for studies with C. elegans on the genetic control of animal development. Another hot area of current research with C. elegans is animal behavior. These nematodes exhibit a wide array of behaviors that can be monitored in the laboratory. One well characterized behavior is chemotaxis. Basically, chemotaxis is movement toward or away from a chemical. More specifically, it involves a change in direction of movement in response to a gradient of a chemical. Positive chemotaxis is movement towards higher concentrations of the chemical; negative chemotaxis is movement towards lower concentrations of the chemical. Several chemicals have been identified that elicit a positive or negative chemotaxis response in C. elegans. To identify the genes involved in chemotaxis, several mutant strains of C. elegans have been generated that are defective for this behavior. Typically these mutant nematodes fail to change direction of movement in response to the chemical gradient. Some of the mutant strains are highly specific to one type of chemical. Other mutants are defective in response to a wide range of chemicals. These genes have been found to encode a wide variety of proteins, including olfactory receptors, transcription factors and neuron secretory proteins. A compound that elicits an unusual and complex chemotaxis response is benzaldehyde. In a gradient of high concentrations of benzaldehyde, C. elegans initially shows a positive chemotaxis response and moves in the direction of higher concentrations. This positive response lasts approximately 20 – 30 minutes. At that point, the behavior of the nematode reverses and it shows a negative chemotaxis response and moves in the direction of lower concentrations. This change in behavior is often cited as an example of simple learning in C. elegans. In this laboratory exercise, you will use chemotaxis mutants to “genetically dissect” the complex behavior elicited by benzaldehyde. Specifically you will be investigating whether the same genes required for the positive chemotaxic response to benzaldehyde are required for the negative response to benzaldehyde. Specific Goals Observe chemotaxis response to benzaldehyde using a standard chemotaxis assay. Compare the chemotaxis behavior of wildtype nematodes with mutant nematodes to analyze whether the same genes are required for both the positive and negative chemotaxic response to this compound. Appreciate how mutants can be used to genetically dissect a complex biological process. Chemotaxis Assay Several simple assays have been used to measure chemotaxis responses. In this exercise we will assay chemotaxis in a Petri dish. A simple test arena is setup in agar Petri plate (Figure 1). Evenly spaced lines drawn on the bottom of the plate are used to divide the plate into 5 sectors (a-e). At one end of the plate (sector a) a small amount of the compound to be tested is spotted. At the opposite end (sector e), a control compound may be spotted (usually water). Nematodes are placed in the center of the plate (sector c) and allowed to move around for one hour. If they have a positive chemotaxis response to the test compound they will move from the center of the plate towards the test compound. If they have a negative chemotaxis response to a compound they will move away from the test spot and end up near the water spot. To observe the dynamic behavior of the nematodes in response to benzaldehyde, they must be monitored every 10 minutes. To quantify the kinetics of movement in response to a compound the number of worms in each sector is determined. A chemotaxis index (WCI) is calculated. [2(# in a) + (# in b)] – [2(# in e) + (# in d)] WCI = (total # of nematodes on the plate) Exercise Obtain 2 chemotaxis plates, rulers and sharpies from instructor. Draw lines on the bottom of the chemotaxis plates dividing it into the five sectors. Label the sectors a-e. Obtain two cultures of C. elegans. One culture of wildtype worms (N2) and a culture of worms defective for a gene required for positive chemotaxis to benzaldehyde (strain 2065 odr-1 gene or strain 2304 odr-2 gene). Rinse worms off plate: Use a P1000 micropipette to add 2 ml of S-buffer to surface of plate. Briefly swirl the water around the plate. Tilt the plate so that the buffer runs to one edge. Use P1000pipette with a wide mouth tip to transfer the worms to a small assay tube. Allow the worms to settle to the bottom of the tube for about 3 minutes. Use the micropipette with the wide tip to remove the buffer above the settled worms. Use the P1000 micropipette with the wide tip to transfer the remaining buffer plus settled nematodes to one edge of the chemotaxis plate within sector c. Tilt the plate slightly so that the buffer containing the nematodes runs to the other edge of sector c. Use the twisted end of a kimwipe paper to draw excess water off of the nematodes. Break up any clumps of nematodes by touching the clump with the tip of the twisted kimwipe. Use the micropipettor to spot 2 µl of 100% benzaldehyde on one side of the plate (sector a). Keep the lid on the plate for the remainder of the experiment. Count the total number of nematodes in each sector of the plate. Record your data. Ignore worms that have failed to move from the location that they were added. They may be dead or injured. Count only the adult hermaphrodites and ignore any young larval stage nematodes. Repeat step 8 every 10 minutes for a total of 90 minutes. Calculate WCI for each 10 min period of the experiment. Graph WCI data for wildtype and mutant nematodes. Lab Report Graph WCI data for wildtype and one strain of mutant nematodes. Submit properly labeled graphs. The goal of this experiment was to distinguish between two hypotheses. Hypothesis I: The positive and the negative chemotaxic response to benzaldehyde used the same genetic pathway (require the same gene products). Hypothesis II: The positive and the negative chemotaxic response to benzaldehyde are different genetic pathways and each requires unique gene products. Write a paragraph analyzing your data with regard to these two hypotheses.