15 points total Biol203 Fall 2013
Homework 11:
Also due in your lab starting Dec 9th and will be graded by your Lab TA.
Your answers should be written either in bullet points rather than sentences
or as a flow chart.
For each question, please write a concluding sentence after your bullet points or flow chart.
You have just identified a lobster cDNA that is 70% identical in nucleotide sequence to the mouse Pax6 cDNA.
1) What bioinformatics steps would you take to show that the gene products have similar function?
Look at coding sequence and determine amino acid homology. If 100% identical at protein level, then proteins have the same function. If Homeodomain stretch of 61 amino acids is identical, then it is likely that proteins have identical function.
2) What molecular biology experiments (not counting Tg or Gene-targeting experiments) would you take to show that the gene products have similar function?
Anything where DNA binding domain and Activator domain would be tested.
The simplest example is a two hybrid experiment with gal4 where the lobster and mouse pax6 domains would be tested in vitro. In this case the DNA target would have the same sequence specificity. Gel shift experiments could be done, but only test DNA binding (2.5pts only).
3) What Transgenic and Gene-targeting experiments would you take to show that the gene products have similar function?
Knockin experiments.
Replace the mouse pax6 coding sequence with that of the lobster pax6 coding sequence. If same phenotypes result in animals, then proteins likely have the same function. In such experiments, students should not be replacing the UTRs within gene-targeted mice.
Otherwise, a knockout of pax6 could be generated and rescued by a transgene with eye promoters/enhancers expressing either the mouse pax6 cDNA or the lobster pax6 cDNA. If the rescue experiments give equal phenotypes in the mouse, then proteins have equivalent functions.
No points for trying to make gene-targeted lobster or any manipulations in the lobster!