Note that some of these questions may require information on complementation from lecture 19 (pages 170-172 and lecture 19 notes).
1. What was the earliest evidence suggesting the possibility of a one gene - one enzyme relationship. Roughly when was this evidence obtained?
2. What is an auxotrophic mutation?
3. A collection of auxotrophic strains of Neurospora that all require tryptophan for growth has been found to involve mutations at a number of different genetic loci. Explain how mutations in different genes can lead to the same auxotrophic growth requirement.
4. In question 3, how would you determine how many different genes were mutated in the collection of mutant strains that were auxotrophic for tryptophan? Include in your answer the results you would expect to see if two strains both contained mutations in the same gene, as well as the results you would expect to see if the two strains contained mutations in different genes.
5. The biochemical pathways involved in tryptophan biosynthesis in E. coli are known. If you had the collection of Neurospora mutant strains described in question 3, how could you use it to determine whether the pathway was the same in both species?
6. You are studying a series of tryptophan auxotrophs that are unable to convert indoleglycerol phosphate to tryptophan. (Assume for purposes of this question that only one enzyme is involved in that conversion).
a. How would you determine whether two such mutations have defects in the same protein coding sequence?
b. If your test in part a. indicates that both mutations involve the same gene, how would you determine whether they involve different sites within that gene?
c. You find two tryptophan auxotrophs that complement each other despite both being unable to utilize indoleglycerol phosphate. What possible interpretations can you give to that observation?
d. Explain how you would distinguish among the alternatives that you proposed in part c.
e. You find that one of your auxotrophic mutant strains fails to form prototrophic recombinants with any of the mutations in one of the complementation groups you are studying. How would you explain that result?
7. You are studying two different mutations that destroy the activity of an enzyme. Complementation analysis reveals that the mutations complement each other. However, when you prepare highly purified enzyme and subject it to SDS polyacrylamide gel analysis under reducing conditions (a procedure that fully denatures the proteins and separates the subunits), you can find only one type of protein. In addition, conventional genetic analysis says that the two mutations are very tightly linked. How can these results be explained. Cite an example that we have studied of such a phenomenon.
8. What is a suicide substrate and how can it be used to screen for auxotrophic mutations?
9. How can complementation studies be done with haploid organisms? (Several answers are possible for various species.)
10. Explain why complementation analysis cannot be done with dominant mutations.
11. Mutations A and B do not complement each other when tested in a recombinant-deficient strain of bacteria. However, in strains that support recombination, there is occassional restoration of function. Explain how this can happen.
12. Two mutant strains (M1 and M2) fail to complement each other. DNA sequence analysis shows that M1 has a mutation in the coding sequence for enzyme E that remders the protein totallly nonfunctional. However, the coding sequence for enzyme E is perfectly normal in M2. What possible explanations can you suggest for this observation? (Hint: think about other ways in which M2 could fail to produce enzyme E).
13. Does complementation between two mutant strains prove that both mutations have occurred within the same biochemical pathway? Explain your answer.
14. In a collection of strains auxotrophic for the same nutrient, does complementation prove that two mutations affect different steps in the biosynthesis of that nutrient? Explain your answer.
15. You have a collection of mutant strains that all fail to make the same enzyme. One of the strains fails to make any mRNA for the enzyme. Without resorting to sequence analysis (which has not yet been covered in this course, how could you determine whether it was a promoter-defective mutation or a deletion of the entire coding sequence?
16. What is meant by the term "heterozygote advantage"? Would you classify this as a form of complementation?
17. Beadle and Tatum proposed a "one gene-one enzyme" theory, based on their early studies of biochemical genetics and complementation in Neurospora. How would you modify that description to take into account more recent data?
18. Prior to DNA sequencing and our current understanding of the genetic code, what evidence was there for the colinearity of gene structure and protein amino acid sequences?
16. What is a prion and what makes the concept of prions so controversial?
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