Announcement

TA review session

Basic method of inquiry in biology

Biological process development, cell division, migration, apoptosis, etc Gene Function Transcription factors, Kinases, phosphotase, ATPase, etc.

Discovery requires an integrated approach

Week 2-4 Week 8-10

Cloning of DNA fragment Northern blot of Rb Gene

GST-pull down Co-immunoprecipitation

Rb is frequently mutated in cancer whose function is to physically interact and inhibit E2F transcription factors, which control cell cycle progression.

Promoter reporter assay RTq-PCR FACS analysis

Week 5

Week 6

final exam

April 29 6:00 pm at TBD.

1. The final is comprehensive and problem solving-based 2. the final is composed of 4 essay questions
25 points each, 100 points in total each essay question is composed of 3 or 4 problems to be solved

3. Give short and concise answers and use flowcharts.

1. For your PhD you join a Drosophila laboratory and embark on a study examining the development of neural connections between the left and right side of the fly. In a novel screen where you picked mutant flies that were unable to clean coloured powder off their backs, you identify five genes that you name pigpen or PIP1-5. Of particular interest to you is the PIP1 mutant. The defect you observed in this mutant is that nerves that normally cross the dorsal midline to make left-right connections instead run parallel to each other on the left side of the back of the fly. Cloning of PIP1 reveals that it encodes a trans-membrane receptor-like protein with an N-terminal signal peptide. (a) To study the expression of the gene you make a promoter-GUS fusion and discover that PIP1 is specifically transcribed in a certain set of dorsal neurons starting very early in development. With great pride, you send your findings off to The Journal of Great Renown. A month later, you stare in dismay at a rejection letter, asking you to provide proof of the expression pattern of your gene. Explain why your paper was rejected and describe an experiment you could perform to satisfy the reviewers. b) PIP has an ortholog (e-value = e -200) in yeast called YIP, which you discovered doing sequence analysis one night. Describe the method you used to find the yeast ortholog and what the e value implies. (c) You discover that the PIP transcripts are expressed at normal levels in PIP1 and PIP2 mutants. Based on your analysis of the mutations present in each mutant, you hypothesize that in pip1, decreased protein stability causes the pigpen phenotype, and in pip2, a defect in the proper sub-cellular localization of the encoded protein is responsible for the pigpen phenotype. Describe experiments you could use to test the hypothesis, and the expected outcomes.

1. For your PhD you join a Drosophila laboratory and embark on a study examining the development of neural connections between the left and right side of the fly. In a novel screen where you picked mutant flies that were unable to clean coloured powder off their backs, you identify five genes that you name pigpen or PIP1-5. Of particular interest to you is the PIP1 mutant. The defect you observed in this mutant is that nerves that normally cross the dorsal midline to make left-right connections instead run parallel to each other on the left side of the back of the fly. Cloning of PIP1 reveals that it encodes a trans-membrane receptor-like protein with an N-terminal signal peptide. (a) To study the expression of the gene you make a promoter-GUS fusion and discover that PIP1 is specifically transcribed in a certain set of dorsal neurons starting very early in development. With great pride, you send your findings off to The Journal of Great Renown. A month later, you stare in dismay at a rejection letter, asking you to provide proof of the expression pattern of your gene. Explain why your paper was rejected and describe an experiment you could perform to satisfy the reviewers.

1. For your PhD you join a Drosophila laboratory and embark on a study examining the development of neural connections between the left and right side of the fly. In a novel screen where you picked mutant flies that were unable to clean coloured powder off their backs, you identify five genes that you name pigpen or PIP1-5. Of particular interest to you is the PIP1 mutant. The defect you observed in this mutant is that nerves that normally cross the dorsal midline to make left-right connections instead run parallel to each other on the left side of the back of the fly. Cloning of PIP1 reveals that it encodes a trans-membrane receptor-like protein with an N-terminal signal peptide. b) PIP has an ortholog (e-value = e -200) in yeast called YIP, which you discovered doing sequence analysis one night. Describe the method you used to find the yeast ortholog and what the e value implies.

1. For your PhD you join a Drosophila laboratory and embark on a study examining the development of neural connections between the left and right side of the fly. In a novel screen where you picked mutant flies that were unable to clean coloured powder off their backs, you identify five genes that you name pigpen or PIP1-5. Of particular interest to you is the PIP1 mutant. The defect you observed in this mutant is that nerves that normally cross the dorsal midline to make left-right connections instead run parallel to each other on the left side of the back of the fly. Cloning of PIP1 reveals that it encodes a trans-membrane receptor-like protein with an N-terminal signal peptide. (c) You discover that the PIP transcripts are expressed at normal levels in PIP1 and PIP2 mutants. Based on your analysis of the mutations present in each mutant, you hypothesize that in pip1, decreased protein stability causes the pigpen phenotype, and in pip2, a defect in the proper sub-cellular localization of the encoded protein is responsible for the pigpen phenotype. Describe experiments you could use to test the hypothesis, and the expected outcomes.

4. Ivermec8n is a commonly-used drug for the medical and veterinary preven8on of gastrointes8nal nematodes. In order to understand the biology of ivermec8n toxicity in nematodes, with the aim to develop new drugs, you decide to do a gene8c screen on a chemically mutagenized popula8on of C. elegans worms to nd mutants that are resistant to ivermec8n. In your screen, you iden8fy mutants aected in 10 genes. You decide to focus on your most resistant mutant, which you have named invulnerable (inv). (a) [5 points] Mapping of INV revealed that it is located in a 40kb region that contains 6 genes. Briey describe two experiments you can do to narrow down the candidate genes responsible for the inv mutant. (b) [10 points] Cloning of INV revealed that it encodes a transcrip8on factor that contains mul8ple copies of transcrip8on factor protein domains from the Zinc-nger family. Some genes containing Zinc-nger domains are alterna9vely spliced. Explain what this is, why it might be important in this case, and how you would go about checking for it, using bioinforma8c techniques. (c) [10 points] Many members of the Zinc nger family are known to be localized to the cytoplasm and imported into the nucleus to regulate gene transcrip8on. You believe that INV should also be seen in both cytoplasm and nucleus. More importantly, you think that the INV protein might be a molecular target of ivermec8n, in that the drug could inhibit the rate of INV import to the nucleus. Describe an approach that you could take to examine if ivermec8n slows INV protein import into the nucleus, including controls, predicted results and basic conclusions.

4. Ivermec8n is a commonly-used drug for the medical and veterinary preven8on of gastrointes8nal nematodes. In order to understand the biology of ivermec8n toxicity in nematodes, with the aim to develop new drugs, you decide to do a gene8c screen on a chemically mutagenized popula8on of C. elegans worms to nd mutants that are resistant to ivermec8n. In your screen, you iden8fy mutants aected in 10 genes. You decide to focus on your most resistant mutant, which you have named invulnerable (inv). (a) [5 points] Mapping of INV revealed that it is located in a 40kb region that contains 6 genes. Briey describe two experiments you can do to narrow down the candidate genes responsible for the inv mutant.

4. Ivermec8n is a commonly-used drug for the medical and veterinary preven8on of gastrointes8nal nematodes. In order to understand the biology of ivermec8n toxicity in nematodes, with the aim to develop new drugs, you decide to do a gene8c screen on a chemically mutagenized popula8on of C. elegans worms to nd mutants that are resistant to ivermec8n. In your screen, you iden8fy mutants aected in 10 genes. You decide to focus on your most resistant mutant, which you have named invulnerable (inv). (b) [10 points] Cloning of INV revealed that it encodes a transcrip8on factor that contains mul8ple copies of transcrip8on factor protein domains from the Zinc-nger family. Some genes containing Zinc-nger domains are alterna9vely spliced. Explain what this is, why it might be important in this case, and how you would go about checking for it, using bioinforma8c techniques.

4. Ivermec8n is a commonly-used drug for the medical and veterinary preven8on of gastrointes8nal nematodes. In order to understand the biology of ivermec8n toxicity in nematodes, with the aim to develop new drugs, you decide to do a gene8c screen on a chemically mutagenized popula8on of C. elegans worms to nd mutants that are resistant to ivermec8n. In your screen, you iden8fy mutants aected in 10 genes. You decide to focus on your most resistant mutant, which you have named invulnerable (inv). (c) [10 points] Many members of the Zinc nger family are known to be localized to the cytoplasm and imported into the nucleus to regulate gene transcrip8on. You believe that INV should also be seen in both cytoplasm and nucleus. More importantly, you think that the INV protein might be a molecular target of ivermec8n, in that the drug could inhibit the rate of INV import to the nucleus. Describe an approach that you could take to examine if ivermec8n slows INV protein import into the nucleus, including controls, predicted results and basic conclusions.