Questions about Brenner (1974)
I. *EMS mutagenesis: What are the most common types of mutations caused by EMS? For this type of mutation, what codons would be mutated by EMS in a single base change and what would they now encode? Are there any codons that cannot be mutated by EMS? Are there any codons that cannot be made by EMS mutagenesis? What amino acids do they encode? Can the most common action of EMS mutate a stop codon away?

II. *M and S sets: What is the difference in the procedure to obtain these two sets of mutations? Why did Brenner use these two different schemes?

III. Other genetic schemes: In both the M and S mutageneses, the mutants were obtained in the F2 generation. Devise schemes to obtain the following mutations and indicate what generation needs to be examined.
A. Dominant mutations.
B. Additional alleles of gene m on an autosome, where you already have one recessive mutation that has been mapped between genes a and b (so you have animals that are homozygous amb).
C. Recessive maternal effect mutations.
D. What screen, not so far given in this problem, can produce animals with mutant phenotypes in the F1 generation?

IV. Brenner’s genes (Table 4). The genes identified have been a gold mine for subsequent researchers. One of the exciting aspects of genetics is that the nature of the genes obtained through a mutagenesis leads one into new areas of biology. Most of the genes identified by Brenner have now been cloned. Look up the genes for each of the following classes in Wormbase and derive a hypothesis about how the genes of give the particular phenotypes they do [the categories of unc genes come from J. Hodgkin (1983) Male phenotypes and mating efficiency in Caenorhabditis elegans. Genetics 103: 43-64.]
A. bli genes (Bli phenotype: blistered cuticle in adults)
B. dpy genes (you don’t have to look at all, just a subset; Dpy phenotype: animals are shorter but as thick as wild type)
C. sma genes (Sma phenotype: animals is proportionately shorter in length and width)
D. Limp paralyzed unc genes (unc-15, unc-36, unc-45, unc-52, unc54, unc-60)
E. Backward unc (unc-4, also unc-55 and unc-59)
F. Forward unc (unc-1, unc-7, unc-9, unc-23, unc-24)
G. Shrinker unc (unc-25, unc-30, unc-43, unc-46, unc-49)
H. tmr genes [tmr-1(now called lev-1), unc-29, unc-38, unc-63)

V. *Incorrect Assignments: We now know that several mutations identified by Brenner as alleles of different genes are alleles of the same gene. By looking at the phenotypes on WormBase, provide a reasonable hypothesis to explain these errors.
A. dpy-2 and rol-2
B. dpy-13 and dpy-16
C. unc-7 and unc-12
D. unc-8 and unc-28
E. unc-18 and unc-19
F. unc-21, unc-29, and unc-56
G. unc-60 and unc-66

VI. "Lethal mutants" (p. 89): Be able to explain this experiment. Start by diagramming all of the crosses. Why is R considered a "crude ratio"? What is the derivation of the formula for the frequency of induced lethals per X chromosomes? What is the frequency?

VII. Mutation rate: How does Brenner calculate the average rate of mutation for autosomal genes? Why did he use the numbers that he did? Was the number “77” correct?

VIII. Saturation: Geneticists like to estimate the number of genes that can be mutated to give a particular phenotype. A screen or selection is saturated if mutations have been found in all (or nearly all) genes that can yield a particular phenotype. For example, Brenner states, "Nevertheless, the results strongly suggest that for the level of phenotype recognition used, the spectrum must be nearly saturated" (p. 92). Consider the following questions to determine if you agree with this statement.
A. An intuitive feeling for whether saturation has been reach is to look at how many alleles have been found for each gene. To take a somewhat silly example imagine that your grade for this course depended on an archery contest in which you were asked to shoot arrows over a barrier to hit an unseen number of targets. You are only told whether you hit a target and if it had been hit before. You “pass” the course when you can say that you have hit all the targets.
1. After shooting several hundred arrows you find that you have hit three targets, one time each. Should you stop and give an answer of three? Why?
2. You continue shooting and after a while find that you have single arrows in four targets and two or more arrows in five targets. Should you stop and give an answer of nine? Why?
3. After an interminable amount of time (during which you greatly resent taking the course) you find that you have hit twelve targets, all of which have at least six arrows in them. Is it now time to stop? Why?

4. The answers you have given make assumptions about the nature of the targets and their accessibility. What are those assumptions? How could those assumptions not be met?
B. Using the same reasoning as in A and the data in Table 4, determine how many lon genes did Brenner identify by mutation and how many alleles of each lon gene were found. Do these results suggest saturation of his mutageneses as he claims?
C. Do the same analysis for the rol genes. Does this analysis give the same result?
D. How do you reconcile the results in B and C, since both are describing the same set of mutageneses?
E. Later in the paper Brenner says that "there is only one gene in which mutations occur to produce twitching animals." However, single mutations have been found subsequently in two other genes (lev-11 and unc-54) that produce twitchers (other mutations in these genes do not give this phenotype). Do these data invalidate Brenner's statement about saturation?

IX. Predicting gene number: Read the handout “Predicting gene number” and answer the following questions using the Poisson analysis described there.
A. After several months of taking my daily change and tossing it in a drawer, I find that I have the following quarters out of a total of 201 quarters (quarters have different backs to them; some with the names of states).

Eagle-back Quarters                   100
States represented by 1 quarter AL, AZ, GA, KY, LA, MO, MD, NV, OH, OK, OR, PA,
                                                   SD, TN
States represented by 2 quarters AK, AR, CA, ID, IN, MD, MA, MN, MT, NH, NM, WY
States represented by 3 quarters CO, DE, MS, NY, RI, VT, WV
States represented by 4 quarters CT, NE, SC, ND
States represented by 5 quarters NC
States represented by 6 quarters WA
States represented by 7 quarters VA
Other quarters                               4 Bicentennial (1976)
                                                      2 Great Smokey Mountains
                                                      1 White Mountains
                                                      1 District of Columbia
Some of the above state quarters CT, GA, MD (2), MN, NM, NC(2), PA, RI (2), SC
were from the Denver (D) mint;       (2), TN, VT,VA (2), WA (1)
the rest were from the
Philadelphia (P) mint.

1. Use the data to estimate how many US states exist.
2. Equal numbers of quarters were made by the two mints. Why do you suspect that there are many more P quarters than D quarters?
3. How does these data serve as an analogy of a mutagenesis?
B. Use the data in Brenner Table 10 to calculate the number of autosomal genes that could be expected to give a visible phenotype. (Note: use the information in V above to correct the data in Table 10.) How far from saturation was he?

X. Number of genes in the genome: How does Brenner calculate the total number of genes in the genome? Why did he use lethal genes for this estimate? Why was this number of genes he obtained (as well as the similar number obtained from work with Drosophila melanogaster) considered a problem at the time this paper was written? What assumption did he make about the nature of eukaryotic genes? Given that we now know that the number of sterile genes, which he did not identify, is approximately that of the lethal genes, do we still have a problem? What do we know now about the structure of eukaryotic genes that might explain these results? The average size of a C. elegans gene is 5 kb and the genome is now known to contain 100 Mb of DNA. Do the number work now? What conclusions do you draw from these data?

XI. Lannate resistance: In describing his results with lannate resistance Brenner (p. 90) says that the resistant strains all have mutations that fail to complement two alleles of unc-17 (e113 and e464). Animals with the e464 mutation are lannate resistant and look like the other lannate resistant mutants. Animals with the e113 mutation, however, do not, they are healthier, larger (the others are smaller), and are not resistant to lannate (i.e., it kills them). All the mutations, however, cause the same type of uncoordination and they all fail to complement each other with regard to the Unc phenotype. We now know that the e464 mutation affects the coding sequence of unc-17, whereas e113 is in the promoter to the gene. How can you explain different phenotype of the e113 animals? (The “gene” itself has a very interesting structure that is in fact conserved in flies and mammals. You might look it up in WormBase.)

Questions about Brenner (1974) I. *EMS mutagenesis: What are the m...