7.013 Quiz 2ANSWERSQuestionValueScore114216316424517613100MIT Department of Biology7.013: Introductory Biology - Spring 2005Instructors: Professor Hazel Sive, Professor Tyler Jacks, Dr. Claudette GardelQuestion 1 a) What role does the acrosome have in fertilization? Circle all that apply. 3 pts i) It responds to egg’s chemoattractants. ii) It has enzymes that digest the zona pellucida. iii) It has enzymes that digest the nuclear membrane. iv) It is the part of the egg that prevents sperm from binding. v) None of the above. b) If calcium is injected into an unfertilized sea urchin egg, what would happen? Circle one. 3 points i) Capacitation would occur. ii) A slow block to polyspermy would be induced. iii) The egg will become fertilized without sperm. iv) The egg will become female. v) The sperm will undergo an acrosomal reaction. c) If we now mix sea urchin sperm with these eggs (in b), will fertilization occur? Circle one. 3 points i) No, capacitation has already occurred. ii) No, the sperm can no longer attach to the egg. iii) Yes, the egg is now in “ready state”. iv) No, the egg is already fertilized. v) None of the above. d) Could a human sperm fertilize a hamster egg? Explain your answer briefly. 5 points No, the egg is protected by a thick zona pellucida that can be penetrated only if the ZP3 ligands within can be bound by the sperm with the appropriate receptor. These interactions are species specific. This triggers the acrosomal reaction which spills out hydrolytic enzymes to make a passage of the sperm through the Zona Pellucida. 2I3 Name______________________________ Question 2 You wish to make your pet glow in the dark so your first step is to clone gfpgene, (encoding green fluorescent protein), downstream of the “ON” promoter which is always active, expressing downstream genes. You have a vector, the PON plasmid, and you’ve isolated a DNA fragment containing a promoter-less gfpgene as well as the tetr gene conferring resistance to tetracycline. The vector carries the lacZ gene encoding ß-Galactosidase which converts the substrate, X-gal, into a blue colored product. It also carries two antibiotic resistance genes: kanr and ampr which r rallow for growth on kanamycin and ampicillin, respectively. The lacZ, kan , and amp geneshave their own promoters (not shown). Assume all restriction sites are unique unless shown otherwise. Assume that only similarly cut DNA ends can ligate with each other. You may use more than one enzyme for your cloning. Assume all digestions are complete (no partial digestions). FragmentSacI NotI XhoI ampr ori SacI SalI EcoRI PON kanr gfp tetr NotI I SalI EcoRV lac Z NotI SalI EcoRV EcoRI PtetPON Plasmid EcoRV Sac I Nota) In order to clone gfpin the correct orientation downstream of PON... i) what enzyme(s) would you cut the vector with? 6 points per side (Eco R I-EcoRI –3 pts) (Sac I-Sac I-3 points) Eco RI EcoRV NotI Sac I SalI XhoI ii) what enzyme(s) would cut the fragment with?Eco RI EcoRV NotI Sac I SalI XhoI b) To select for the desired construct with insert you should plate the ligation on medium with...Circle all that apply. 3 points Ampicillin Chloramphenicol Kanamycin Tetracycline X-Gal c) Bacteria transformed with original vector would grow on medium with...Circle all that apply. 4 points Ampicillin Chloramphenicol Kanamycin Tetracycline X-Gal d) A fellow student cloned the gfpinsert into the vector using NotI. However he found that there was no expression of gfp. Why? 3 points The promoter PON would be removed. 3Question 3 You want to identify genes that control muscle development. Zebrafish are used to study vertebrate development, because the embryos are transparent, abundant and easily harvested. You have two temperature sensitive mutant fish that have mutations in unknown genes causing interesting phenotypes you name “Limp” and “Paralyzed”. At 23°, these mutant fish eggs develop normally but if they are shifted to the restrictive temperature, 30°, within 5 hours after fertilization, the embryos exhibit the limp or paralyzed phenotypes. You grow the wildtype and mutant embryos at 23°C, and then shift them to 30° at 4 hours after fertilization. At hour 10, you make sections through the trunk of the embryos and identify different cell types that should be present at this stage in development with specific dyes. FP stained by FloorPlate dye M stained by Muscle dye N stained by Notochord dye Wild type Limp Paralyzed N FP M M NN FP F P F P a) The Limp and Paralyzed mutants could be defective in.... (Circle all that apply.) 5 points i) the production of a protein. ii) the splicing of an mRNA. iii) responding to cell nonautonomous signals. iv) post translational modification of a protein. v) the transcription of a gene. b) You grow the wildtype and the mutant fish embryos at 23°C and then shift them to 30° at 6 hours after fertilization. At hour 10, you stain as before. You observe that the mutant strains stain exactly like the wildtype. Why is this? Circle all that could be true. 3 points i) Transcription factors had already induced necessary genes. ii) Enough cell specific protein had been made at 23°C. iii) Temperature sensitive proteins are no longer required by hour 6. iv) Temperature sensitive proteins are required until hour 10. v) None of the above. 4Name______________________________ You take Limp mutant fish embryos and transplant in wild type donor cells that have been marked with a Fluorescent dye (gray). You then section the trunk and again stain as above. N FP F P MN FP F P F P N FP M M marked cells derived from WT marked cells derived from WT marked cells derived from WT notochord precursor cells floorplate precursor cells muscle precursor cells c) These results suggest that the Limp mutation is ... (Not Graded) Cell autonomous Cell nonautonomous d) Briefly explain your answer in e). (Not Graded because of printing error.) 2 free points. Wild type Notocord can send a signal to cells with receptors to become muscle. You repeat the above experiments with Paralyzed mutant fish embryos and transplant in wild type donor cells that have been marked with a Fluorescent dye (gray). You then section the trunk and again stain as above. N MN FPN marked cells derived from WT marked cells derived from WT marked cells derived from WT notochord precursor cells floorplate precursor cells muscle precursor cells f) These results suggest that the paralyzed mutation is ... 2 points Cell
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