Problem Set 5

Due Thursday Feb 16 before 1 AM. You may submit as a group. 1. (1) In experiments on Drosophila patterning, you treat fertilized eggs with a drug that completely blocks RNA polymerase II, which is required for all mRNA synthesis. Explain briefly if and how you would expect this treatment to affect the process of initial anterior-posterior patterning of the embryo. Ans probably not much. The primary genes required for patterning are maternally transcribed the egg polarity genes bicoid (anterior) and nanos (posterior), and the gap genes hunchback and caudal. Both of the egg polarity genes affect translation of the gap genes, but the lack of RNA pol II would not affect translation. . 2. (2) You are studying a gene in Drosophila called no antennae (nat). Flies homozygous for a nat loss-of-function mutation have no antennae. a. (.5) what does the nat gene product normally do in the fly embryo it must be responsible for initiating antennae development, since when it is gone, no antennae develop. b. (1.5) You do not yet know exactly what nat does, or how important it is during fly development. To figure this out, you want to overexpress the nat transcript in a novel (ectopic) location, and see what happens to those cells. Describe how you could do this experiment, and predict what you might see as an outcome. (1 point) Two possible ways. 1. use a simple transgene, where you introduce a construct that has a eye gene promoter hooked up to the nat coding sequence. Generate transgenic flies with this transgene, observe what happens to cells in the eye, which should now be expressing nat. 2. Use the GAL-4 UAS system. Generate two transgenic flies, one carrying a gene for the transcription factor GAL-4, driven by a promoter specific to the eye imaginal disc,and the other carrying a transgene comprised of the UAS sequence upstream of the nat coding sequence. (.5 pt) If you hypothesize that the gene might be involved in regulating antennae development, you might expect to see antennae like structures either in place of the eye or in addition to the eye. Note, mouse techniques like homologous recombination and cre-lox cannot work in Drosophila.) 3. (4 pts) Both Notch and Delta loss of function mutations (when homozygous) lead to the presence of bristles (a neuronal cell type) in cells that normally make cuticle (epidermis). For an exercise in your lab course, your TA has produced two strains of flies with small mosaic patches--one strain has patches of Notch mutant cells the other strain has patches of Delta mutant cells in their epidermis. The mosaics have been made using a linked color marker, yellow, so that the homozygous mutant cells are a different color than the surrounding wild type cells (black). Each student has been given a different mosaic fly as an unknown and asked to identify it as a Notch or a Delta mosaic. a) (2) a) On your fly, you notice that cells in the middle of the yellow patches make extra bristles. However, along the border of the yellow patch, some of the yellow cells are phenotypically normal. Is your fly mosaic for Notch or Delta How would you explain your conclusion to the instructor Delta. The presence of the mutant phenotype in non-mutant cells (darker cuticle) indicates that the effect of the mutation is cell non-autonomous. Therefore, the defective component must be the ligand Delta rather than the receptor Notch. The yellow cells along the border are mutant for Delta, but still have the N receptor, and can receive the Delta ligand from the adjacent cells, thus they appear phenotypically normal. b) (2) Some other students in the lab were given flies mosaic for the other gene. How and why do their flies appear different from yours In the Notch mosaics, only the yellow cells (the homozygous mutant ones) make bristles. This gene acts non-autonomously (it encodes the receptor), so only the mutant cells will have a mutant phenotype. (2 pts) a. (1) What is the difference between a transgenic mouse and a chimeric mouse Explain. A transgenic mouse has a foreign piece of DNA in every cell in its body. A chimeric mouse has two kinds of cellsnormal cells, and cells that have a transgene (or have some other genotype different from the first kind of cell). b. (1) If a chimeric mouse is mated to another chimeric mouse, can you predict what fraction of the offspring will be transgenic Why or why not You cannot, because the fraction that are transgenic has to do with what fraction of the mouses germ cells contain the transgene. If they all do, and the mates germ cells also all contain the transgene, then all the progeny of these mice will be homozygous for the transgene. If only some of each of the mouses germ cells contain the transgene, then some of the mouses offspring could be wild type, some heterozygous for the transgene, and some homozygous.