fushi tarazu is the name given to a Drosophila segmentation gene that when mutant results in a larva with half the number of segments. Translated from Japanese fushi tarazu means exactly this. The gene product of the fushi tarazu gene is expressed in seven bands of cells along the anterior-posterior axis of the blastoderm embryo. These ftz expressing cells are the primordial cells for the segments deleted in the ftz mutant larva. The gene product of the ftz gene can act as a DNA-binding transcription factor.
The structure of the labs is as follows. I have supplied you with a set of protocols as you would have to follow in a real laboratory. You are responsible for figuring out what the experiment is showing and how it works. This is to be done before you step into the lab, by having read the pertinent references listed below--each lab has particular papers assigned to it which will the topic of the quiz. During the first hour of the lab you will be given 5 minutes to answer a question for that lab section. This will occur in Lab 1, 2, 3, 4, 5. Each question is worth 1 mark. All paper below are linked to pdf files.
General (Not subject to the quiz, but required reading)
Hafen, E., Kuroiwa, A., Gehring, W. J. Cell 37, 833-841 (1984).
Caroll, S. B. and Scott, M. P. Cell 43, 47-57 (1985).
Jaynes, J. and O'Farrell, P. H. Nature 336, 744-749 (1988).
Ohkuma, Y., Horikoshi, M., Roeder, R., Desplan, C. Cell 61, 475-484 (1990).
Caroll, S. B. and Scott, M. P. Cell 45, 113-126 (1986).
Laughon, A. and Scott, M. P. Nature 310, 25-31 (1984).
Hiromi, Y. and Gehring, W. J. Cell 43, 603-613 (1985).
Schier, A. F. and Gehring, W. J. PNAS 90, 1450-1454 (1993).
Exp1 (readings)
Weiner, A. J., Scott, M. P., and Kaufman, T. C. Cell 37, 843-851 (1984).
Jürgens, G., Wieshaus, E., Nüsslein-Volhard, C., and Kluding, H. Roux's Arch Dev Biol 193, 283-295 (1984).
Exp2 (readings)
Struhl, G. Nature 318, 677-680 (1985).
Ish-Horovicz, D., Pinchin, S., Ingham, P. W., Gyurkovics, H. Cell 57, 223-232 (1989).
Fitzpatrick, V. D., Percival-Smith, A., Ingles, C. J., and Krause H. Nature 356, 610-612 (1992).
Exp3 (readings)
DiNardo, S., and O'Farrell, P. H. Genes and Dev. 1,1212-1225 (1987).
Howard, K., and Ingham, P. Cell 44, 949-957 (1986).
Exp4 (readings)
Tautz, D. and Pfeifle, C. Chromosoma 98, 81-85 (1989).
Ingham, P. W., Baker, N. E., and Martinez-Arias, A. Nature 331, 73-75 (1988).
Exp5 (readings)
Hiromi, Y. and Gehring, W. J. Cell 50, 963-974 (1987).
Schier, A. F. and Gehring, W. J. Nature 356, 804-807 (1992).
Hyduk D. and Percival-Smith A. Genetics 142, 481-492 (1996)
Guichet, A., Copeland, J. W. R., Erdelyi, M., Hlousek, D., Zavorszky, P., Ho, J., Brown, S., Percival-Smith, A., Krause, H. M. and Ephussi, A. Nature 385, 548-552 (1997)
You should be able to answer the following questions at the beginning of every lab (this may or may not be the question asked during the lab period.
What are we doing today?
How are we doing it?
What has been done before you started the experiments, that is, where did the material (flies) come from and what is special about them?
What are the expected results?
Questions you should be able to answer from your readings. The following list of questions is only a sample of what you should be able to answer from you readings. Being able to answer only the following list of questions is not sufficient.
Exp. 1. What is a balancer chromosome? What are the characteristics of the ftz phenotype? How was ftz first identified? How was the ftz gene isolated?
Exp 2. What are we doing today, and why is it important?
Exp 3. What is the difference between the three fly stocks used? How were the two experimental stocks made? Why is it important for this experiment to use a heat-shock promoter as opposed to a constitutively expressed promoter? What are the individual components that we add during the experiment doing? (may not find this in your papers, so you may have to think, or go to the vector laboratories web site and look up vectastain elite kit).
Exp 4. What is occurring in the individual steps in this experiment?
Exp 5. How do we identify the homozygous ftz embryos independent of the ftz phenotype? What activity of FTZ are we assaying in this experiment? What is X-gal being used for in this experiment? What does the change of specificity experiment show and how? What are you going to show that is interesting relative to the change of specificity experiment?
Exp 1
Monday or Tuesday. You prepare cuticles from the wild-type (OreR), Ki ftz11/TM6B, P{walLy} (ftz11), and ry ftz13/TM3 stocks.
Exp 2
Sunday or Monday. You heat-shock embryos of the fly lines OreR, hsftz 3-413 (245), and hs ftz 274-302 (230).
Monday or Tuesday. You prepare cuticles from the various strains.
Monday or Tuesday. You prepare the embryos for staining with Engrailed antibody and in situ localization of Wingless mRNA. The flies that you will collect the embryos from, will be wild-type ry ftz13/TM3 and Ki ftz11/TM6B, P{walLy} (ftz11). You will collect embryos for 1 hr and age at 25 °C for 41/2 hr. You will then fix the embryos as described in the protocol.
Exp 3 (longest lab of the term)
Monday or Tuesday. This is a long lab that will start early in the morning. Engrailed antibody staining of wild-type and ftz mutant embryos.
Exp 4
Sunday or Monday. Prepare, prehybridize and start hybridization of the embryos with the digoxigenin labeled wingless RNA probe.
Monday or Tuesday. Wash the embryos, add anti DIG antibody, and develop the colour reaction.
Exp 5
Monday or Tuesday. This is a long lab. Collect embryos from DH301 crossed to DH502 and P{ hs ftz 257-316}/CyO, P{hb-LacZ Thb8 w+}; ftz13 ry506 es/ TM3, P{hb-LacZ Thb8 w+} crossed to DH502. Administer a 15 minute heat-shock at 36.5 C at 3:20 hrs AEL. Stain for ß-galactosidase activity at 4:30 AEL. Separate out the darkly staining embryos in the evening. Stain overnight, and mount the embryos on Tuesday.