TA review session Announcement

Gene Function Transcription factors, Kinases, phosphotase, ATPase, etc.

Biological process

development, cell division, migration, apoptosis, etc

Basic method of inquiry in biology

Discovery  requires  an  integrated  approach…  

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

GST-pull down Co-immunoprecipitation

Cloning of DNA fragment Northern blot of Rb Gene

Promoter reporter assay RTq-PCR

3

FACS analysis

Week 2-4 Week 8-10

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  find  mutants  that  are  resistant  to  ivermec8n.  In  your  screen,  you  iden8fy  mutants  affected  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.  Briefly  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-­‐finger  family.  Some  genes  containing  Zinc-­‐finger  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  finger  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  find  mutants  that  are  resistant  to  ivermec8n.  In  your  screen,  you  iden8fy  mutants  affected  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.  Briefly  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  find  mutants  that  are  resistant  to  ivermec8n.  In  your  screen,  you  iden8fy  mutants  affected  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-­‐finger  family.  Some  genes  containing  Zinc-­‐finger  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  find  mutants  that  are  resistant  to  ivermec8n.  In  your  screen,  you  iden8fy  mutants  affected  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  finger  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.