Lecture 6: Molecular basis for Mutation
1. What is meant by a point mutation and how is it distinguished from other types of mutation?
2. What is tautomerization and how is it believed to contribute to mutation? Describe two different types of tautomerization that are known to occur in nucleic acid bases.
3. What is a silent mutation and how could you detect it?
4. Can a missense mutation be phenotypically silent? Explain your answer.
5. What mechanism is usually responsible for a temperature sensitive mutation?
6. Distinguish between the following pairs in a manner that makes it clear that you know what each is and how they differ.
a. Missence mutation and nonsense mutation.
b. Transition and transversion.
c. Positive and negative frameshift mutations.
d. Phenotype and genotype.
e. Reversion and suppression.
7. What effect would you expect reversion caused by a second mutation within the same coding unit (intragenic suppression) to have on the amino acid sequence of a protein that initially loses function as a result of the following types of mutation? Include in each answer the nature of the second mutation that is causing reversal of the mutant phenotype. (You will find it necessary to speculate a bit to answer some of these).
a. Frameshift with the addition of one nucleotide pair.
b. Missense mutation caused by a transition.
c. Missense mutation caused by a transversion
d. Nonsense mutation. (Caution -- do not invoke intergenic suppression here)
e. Frameshift with loss of one nucleotide pair.
8. Mutations are known that lower mutation rates from those encountered in wild type cells. Why do you think evolution has not selected for the lowest possible mutation rates and the greatest possible degree of genetic stability?
9. Distinguish between somatic and germ line mutations in a manner that makes it clear you know what each is and how they differ.
10. How does a suppressor strain of bacteria suppress the effects of a nonsense mutation in a bacteriophage genome? Why would you expect the efficiency of such a suppression to be relatively low?
11. What kinds of mutations are capable of true reversion? What kinds are not?
12. Explain why you would not expect to encounter a temperature-sensitive mutation caused by a frameshift.
13. In boxed example 2.4 (pages 39-41 of textbook) the accumulation of Okazaki fragments was studied in a temperature-sensitive mutation that lacked DNA ligase activity at non-permissive temperatures. Why was it necessary to use a temperature-sensitive mutant strain for this study?
14. Describe three ways in which mutagenic chemicals may cause mutation. What characteristics do the mutagenic chemicals have in each case? What type of mutation would you expect in each case?
15. An auxotrophic bacterial mutant is one that requires a nutrient for growth that is not required by the wild type bacteria. An auxotrophic strain of E. coli is only able to grow when it is supplied with tryptophan in its culture medium. Non-mutant E. coli are capable of synthesizing their own tryptophan and do not require it to be supplied as an external nutrient.
a. Can growth of the mutant strain in a medium containing tryptophan be described as reversion of the mutation? Explain your answer.
b. Can growth of the mutant strain in a medium containing tryptophan be described as suppression of the mutation? Explain your answer.
c. If a large number of the mutant bacteria are plated on nutrient agar that contains no tryptophan, a few colonies of cells that grow well without added tryptophan are sometimes found. What does this tell you about the probable molecular nature of the auxotrophic mutation?
d. Same as part c, except that there is no growth at all without tryptophan.
e. An auxotrophic strain is detected that will not grow without tryptophan under standard culture conditions, but it grows slowly in the absence of added tryptophan when the incubation temperature is lowered. What is the probable molecular nature of the mutation in this strain?
16. Describe the procedures that were first used to demonstrate the colinearity of mutations with the amino acid sequence of proteins. (Hint: go back to boxed example 4.1 on pages 92-93 if you are having trouble with this one).
17. What differences would you expect in mutations caused by intercalating agents, such as acridine orange, as opposed to base analogs, such as 5-bromouracil. Explain the mechanisms that lead to the differences in the mutations caused by these two agents.
18. Which of the following types of mutational mechanism would you expect to generate temperature-sensitive mutants. Explain the reasoning behind your answer in each case.
a. Transition.
b. Transversion.
c. Frameshift.
d. Deletion.
e. Mutations caused by alkylating agents.
19. What mechanism is believed to be responsible for expansion of triplet repeat sequences?
20. Name two human genetic diseases that have been shown to be caused by expansion of triplet repeats.
21. Explain the role played by transfer RNA in the suppression of nonsense mutations.
22. Identification of open reading frames is important aspect of genome sequencing projects.
a. What is an open reading frame?
b. Why would you not expect to encounter open reading frames in DNA (or RNA) that has a truly random sequence?
c. Assume you have access to a high powered computer and help with all needed programming. The complete circular chromosome of E. coli contains 4,639,221 base pairs (table 7.5, page 208). Explain the steps that would be needed to identify all of the open reading frames contained in that chromosome. (Remember that genes can be on either strand of the DNA and that there are three possible reading frames in each direction).
d. What additional features would you look for to try to determine whether each of the open reading frames you detect is actually a functional gene (Hint: think in terms of items covered in the two previous lectures).
e. Would you expect the open reading frame to include the entire sequence that is transcribed for each funcitonal gene? Explain the reasoning behind your answer.
f. What additional complexities would you encounter in an attempt to identify open reading frames in eukaryotic chromosomal DNA sequences?
23. Describe a mutational mechanism that may allow a CG to AT transition to occur without detection by repair systems.
24. You have a collection of nonsense mutations that change the properties of a strain of bacteriophage but do not prevent them from multiplying. A stain of E. coli is found to be capable of suppressing the mutant properties of some of the bacteriophage, but not of others. Speculate on the mechanisms that are responsible for the differences. You should be able to come up with two quite different answers.
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