Exam 1
Inspired by Dominique Belin’s Coursera course “Classical Papers in Molecular Genetics”
Scientific Questions
- The argument by Avery, MacLeod, and McCarty (1944)[1] that DNA carries genetic information rests in part on the assertion that the preparations they used to transform Pneumococcus contain little protein. What is the quantitative basis for that assertion?
3.a. One linchpin in their argument is the measurement of N/P ratios in their preparations. What is the predicted N/P ratio of pure DNA? Don’t rely on a number you may find. Instead, calculate the ratio yourself, basing the calculation on the chemical structure of DNA. Provide the calculation and an explanation of it, perhaps accompanied by the chemical structure you used. Also give the result of your calculation.
3.b. What is the predicted N/P ratio of pure protein? Again base your answer on a calculation, one that relies on the chemical structure of proteins. Provide the calculation and an explanation of it,
3.c. Quantitatively, what is an upper limit for protein contamination in the experiments of Avery, MacLeod, and McCarty? Use data presented in their article and the theoretical N/P ratios you calculated in Questions 3a and 3b to make such an estimate. Provide the calculation, pointing to specific data found in the article, and an explanation of it,
3.d. Suppose that Avery, MacLeod, and McCarty were wrong in concluding that DNA is the transforming principle and protein is the real source (of course their experimental observations remain correct). Suppose further that their preparations were contaminated by protein 0.1% by weight (never mind your answer to Question 3c) and that the active principle is not protein in general but one specific protein present in a 1:1000 ratio of all Pneumococcus proteins. Calculate the number of those specific proteins present in a preparation able to produce one transformant. Point to specific data in the article to support the calculation and present that calculation along with an explanation.
There’s a reason why so many of the articles in the course focused on phage T4. People tend to look for things where the light is best, and the experimental system developed by Seymour Benzer cast a very bright light indeed. The remaining questions are devoted to experiments that make use of the T4 system, and to help you get your hands on it, you will be invited to use a simulation provided in BioBIKE. I know you have had prior experience with BioBIKE, but if it has flitted from your mind, you might want to go through a short hands-on tutorial[2] to remind you of the conventions and pitfalls of the environment. I must stress that this isn’t a course on BioBIKE. The simulations are designed to help you focus on scientific questions. If you find yourself focusing instead on technical problems of how to get BioBIKE to do what it’s supposed to do, do not hesitate to contact me so that I can help you get past the obstacle.
To get to the simulation, start by going to the ViroBIKE[3] instance of BioBIKE and logging in. Then mouse over the INPUT/OUTPUT button and click RUN FILE to bring it into your workspace. Enter the filename “t4-simulation.bike” (in double quotes) and close the box by pressing Enter or Tab. Then mouse over the Options icon and click SHARED. You will see something like this:
A few seconds after executing the function, you should find your function pane adorned with two new blue buttons: Functions and Variables. They will prove valuable in the questions that follow.
- A key advantage provided by the T4 system was the ability to produce and map mutants in the rII region very rapidly. Crick et al (1961)[4] made good use of this facility to map their many rII mutants. In this question you will use the methods of Benzer (1961)[5] to map one of their mutants plus a mystery mutant.
4.a. Crick et al (1961) showed six mutants at the far right of their partial map of rIIB (it is now known[6] that all six are alterations at the same nucleotide). Use the strains and procedures of Benzer (1961) to map one of the named mutations, FC30. You’ll find on your Variables menu a T4 strain (named FC30) carrying the FC30 mutation. You’ll find on your Functions menu a useful function, CROSS, which, when brought down to your workspace, will look like this:
How can you apply Benzer’s methods to map FC30? Provide a diagram that illustrates your strategy (crude is OK). Justify each step according to the characteristics of T4, rII, and E. coli.
4.b. Use the CROSS function and strains available on the Variables menu to map the FC30 mutation. Provide a table that lists each cross you perform and the quantitative results of the crosses, plus an argument for the map position you believe to be correct on the basis of your results.
4.c. Use the CROSS function and strains available on the Variables menu to map Mys1 a mysterious T4 strain given to you on the Variables menu. Provide a table that lists each cross you perform and the quantitative results of the crosses, plus an argument for the map position you believe to be correct on the basis of your results.
- Both Crick et al (1961) and Benzer and Champe (1962)[7] were enamored with a particularly remarkable mutant strain, T4 r1589 (called simply 1589 by Benzer (1961) and appearing on your Variables menu as SB-1589). The deletion mutation borne by this strain fuses rIIA with rIIB and was well used by both groups to draw important inferences that, sadly, we don’t have time to dwell on here. Instead, we’ll focus on the immediate characteristics of the deletion.
I have provided you along with this exam a file containing the DNA sequence of the rII region of bacteriophage T4 (the same sequence with and without coordinates). Make a copy of it and modify it to reflect the r1589 deletion. You don’t have enough information to map the mutation precisely to the sequence, but from the known approximate map position and characteristics of the r1589 strain you can make a pretty reasonable facsimile. Your task will be made easier by the following functions:
You can find these functions from the alphabetical ALL button. Paste sequences you create into the entity box between double quotes and then execute READING-FRAMES-OF.
The second function that might do you some good is shown below:
The inner SEQUENCE-OF rids your sequence of line feeds and such. The outer SEQUENCE-OF displays the translated sequence with coordinates.
Provide the sequence you make, derived from the one in the provided file but with a deletion you think is similar to that present in r1589. Provide also an account of the characteristics you think you captured and evidence that you’ve captured them.
- Now for the main experiment from Crick et al (1961). The first part of their experiment was to create new T4 strains that contained mutations capable of suppressing FC0. Even this single part proceeded in multiple steps, which have been simulated so that you can replicate the experiment yourself. You can read about the tools available to you here,[8] but the main help may be the article itself, Crick et al (1961), and whatever resources you may have to understand it.
Note: Don’t attempt to load “crick-1961-simulation.bike” as described in the documentation. All of the functions you need are already loaded through “t4-simulation.bike”.
6.a. How can you apply these tools to create a strain that has a mutation that suppresses the mutation in FC0 (but does not contain the FC0 mutation)? This is essentially the same question as asking how Crick et al did it. Provide a diagram that illustrates your strategy (crude is OK). Justify each step according to the characteristics of the methods and strains you exploit.
6.b. Use the tools available to create just one of the desired mutant strains (which will not contain the FC0 mutation). Provide a table that lists each step you perform and the quantitative results of each step, plus a brief discussion as to why you believe the strain you ended up with has the appropriate characteristics.
- In a logical tour-de-force, Brenner et al (1965)[9] combined genetic evidence with codon assignments from other labs to identify two nonsense codons. Experiments using hydroxylamine as the mutagen played a major role in their argument. Consider their experiment in which they subjected wild-type bacteriophage T4 to hydroxylamine and classified resulting amber rII mutants in either Set K or Set B.
7.a. Describe how the experiment was conducted. Provide a diagram that illustrates their strategy (crude is OK). Justify each step according to the characteristics of the methods, the strains, the mutagen.
7.b. Return to the sequence of the rII region in the file that accompanied this exam and identify a site that when subjected to hydroxylamine could produce a class K amber mutant. Do the same regarding a class B amber mutant. For the purposes of this question presume that any mutation leading to an amino acid change produces a mutant rII phenotype (but note that not every nucleotide change will do this). Provide the sites (the nucleotide plus 10 nucleotides on either side — coordinates would also be nice). Explain in some detail why the sites you chose have the proper characteristics and would lead to the appropriate mutants. Finally describe what deductions concerning the genetic code can be made from your mutants.
References
[1]. Avery OT, MacLeod CM, McCarty M (1944). Studies on the chemical nature of the substance inducing transformation of Pneumococcal types: Induction of transformation by a desoxyribonucleic acid fraction isolated from Pneumococcus Type III. J Exp Med 79:137-158.
[2]. BioBIKE Syntax and Conventions (http://biobike.csbc.vcu.edu/Doc/externaldf/Syntax-and-conventions.pps)
[3]. ViroBIKE, an instance of BioBIKE. (http://biobike-9003.csbc.vcu.edu/biologin)
[4]. Crick FHC, Barnett L, Brenner S, Watts-Tobin RJ (1961). General nature of the genetic code for proteins. Nature 192:1227-1232.
[5]. Benzer S (1961). On the topography of the genetic fine structure. Proc Natl Acad Sci USA 47:403-415
[6]. Shinedling S, Singer BS, Gayle M, Pribnow D, Jarvis E, Edgar B, Gold L (1987). Sequences and studies of bacteriophage T4 rII mutants. J Mol Biol 195:471-480.
[7]. Benzer S, Champe SP (1962). A change from nonsense to sense in the genetic code. Proc Natl Acad Sci USA 48:1114-1121.
[8]. Documentation for Crick et al (1961) simulation (2012). (http://www.people.vcu.edu/~elhaij/bnfo300/17/Units/Translation/Crick-1961-simulation/crick-1961-simulation.html)
[9]. Brenner S, Stretton AOW, Kaplan S (1965). Genetic code: the ‘nonsense’ triplets for chain termination and their suppression. Nature 206:994-908.
