Bios 344 Molecular Biology and Genetics Study Questions on Recombination Mapping and Restriction Mapping 1. A, B, and C are dominant alleles; a, b, and c are recessive. The cross was made ABC/abc abc/abc. Among 1000 progeny the different phenotypes appeared in the following numbers: ABC 2. 347 Abc 1 abC 8 AbC 145 aBC 2 ABc 9 abc 336 aBc 152 (a) Deduce the map order and calculate the map distances between markers. (b) Do the double crossovers show interference? A test cross between Drosophila females heterozygous for 3 pairs of markers (A/a, B/b, and D/d) and males homozygous for the recessive alleles gave the following results: ABD 23 abd 26 ABd 51 abD 48 AbD 426 aBd 425 Abd 0 aBD 1 How were members of the allelic pairs distributed in the members of the appropriate chromosome pair of the heterozygous females? What is the sequence of these linked genes in their chromosomes? Calculate the map distances between the genes. 3. In Drosophila, assume three pairs of alleles, +/n, +/o, and +/p. Genes n, o and p are all recessives and sex-linked. They occur in the order n-o-p in the X-chromosome, with n being 12 map units from o and o being 10 units from p. The coefficient of coincidence for this region of the X++p chromosome is 0.5. From a cross between females of the genotype no+ and the wild type males, predict the kinds and frequencies of phenotypes that would be expected to occur in a progeny of 2000 individuals. 4. Curved wing and brown eyes are recessive characteristics in Drosophila. The genes for these characteristics are located about 30 units apart in chromosome 2. Suppose that you wish to obtain a double-mutant stock that is curved, brown. You have available for breeding purposes a singlemutant stock having brown eyes and normal wings, and a double-mutant stock which has normal eyes but which is homozygous for curved wings and also homozygous for gene d, a sex-linked recessive mutation. (The starting strains are homozygous at all loci.) Outline the steps you would take in obtaining as efficiently as possible a true-breeding stock for curved wings and brown eyes, which is also homozygous for the wild-type D allele. For each step show the genotypes of the parents to be used in the cross, the genotypes of progeny that will be useful in succeeding steps and the probability of obtaining such progeny. Remember that there is no recombination in male Drosophila. 5. You have two phage mutants, one temperature sensitive and the other suppressor sensitive. You want to measure the frequency of recombination between the two mutations. Under what conditions do you do the recombination? Under what conditions do you measure the frequency of recombinants produced? 6. Suppose that you do a cross between one phage which is a-b-c+ and another which is a+b+c-. You select for recombinants which are a+c+. Either b- or b+ phage can grow under the selective conditions. 7. (a) Among the a+c+ recombinants selected, you find that 0.1% are b+, 99.9% b-. What conclusions can you draw? (b) What additional experiment would permit you to distinguish between the possible conclusions? (c) What conclusions can you draw if most of the a+c+ recombinants are b+? (d) What if the number of b- and b+ were about equal? You have three strains of phage T4, each carrying a single mutation: Strain Mutation 1 a 2 b 3 d In mixed infection with strains 1 and 2, approximately 0.0001 of the progeny are wild-type recombinants. In mixed infection with either 2 and 3 or 1 and 3, approximately 0.01 of the progeny are wild type recombinants. (a) What can you tell about the map positions of the three mutations from the data already given? (b) What further experiments would be the most effective way of determining completely the map positions of these three mutations relative to each other? (Your experiments may include recombinations between the three strains given, to produce new strains. They may not include modification of any of the strains by mutation or introduction of any additional strains; i.e., the only strains that can be used are the original 3 and the strains produced by recombinations between the original 3. (c) Give an example of the sort of data that might be obtained from the experiments in (b) and state the conclusions about map order that you would draw from those data. 8. A | B | 1 C | D | E | 2 F | 3 G | H | 4 I | J | 5 The five lines above represent five T4 chromosomes, each with a deletion defined by the two cross lines. For instance, chromosome 1 has the region between A and B deleted. A point mutation, P, is isolated which forms wild-type recombinants with phage 1, 2, 4 and 5, but not with 3. This implies that the above indicated location is wrong for one of the deletions. You guess that the mistake may be in phage 3, so you test its recombination with the other phages and find that it recombines with 1 and 5 but not with 2 or 4. (a) Was your guess right or wrong? (b) Suggest a change in the location of one of the ends of one of the deletions that would correct the error and make the position of each of the deletions consistent with all of the above data. NOTE: There is more than one possible answer. (c) To test the validity of the change that you suggested in part (b), what cross would you make that would produce no wild-type recombinants if the original order were correct but will produce wild-type recombinants if your suggested change is correct? (d) Assuming that the change that you suggested in (b) is correct, and that no other corrections are necessary, where does the mutation P map? 9. Limit digestion of phage DNA with Eco RI yields 8 fragments A, B, C, D, E, F, G, H. Digestion with Sal I yields 3 fragments, I, II and III. When each of these is digested with Eco RI, the fragments produced are: I. A, C, F and one new fragment II. E, G, and one new fragment III. B, H Partial digestion with Eco RI yields several large fragments. One of them, upon complete digestion with Eco RI, yields fragments B and G. Another yields C, F, and H; and another yields A and F. Construct the map for phage . 10. Suppose that you digest mouse DNA with each of three restriction enzymes: Bgl II, Xba I, and Eco RI. The digests are subjected to electrophoresis and three Southern blots are made, each having all three digests. P32-labeled probes are made from three different restriction fragments, each also obtained from the mouse genome. Two of these are Eco RI fragments J and K, and the third is Xba I fragment P. The three Southern blots are hybridized with the three labeled fragments and autoradiographs are made, resulting in the data shown below. The number next to each band represents the size of the DNA fragment in that band, in kilobases. Blot 1. Probed with Eco RI fragment J: Digest: Bgl II Eco RI Xba I 12.2 10.0 9.6 7.1 6.2 4.8 Blot 2. Probed with Eco RI fragment K: Digest: Bgl II Eco RI Xba I 10.0 8.5 6.9 4.8 3.3 Blot 3. Probed with Xba I fragment P: Digest: Bgl II Eco RI Xba I 10.0 9.6 7.1 6.2 Assume that each fragment shown is the only one of its size generated by digestion of the mouse genome by the enzyme that produced that fragment. Construct a restriction map of the region of the mouse genome defined by these probes. There may be ambiguities in the map, but do it as accurately as the data allow. Bios 344 Molecular Biology and Genetics Answers to Study Questions on Recombination Mapping and Restriction Mapping 1. 2. (a) B (b) yes (a) 30% 10% A a 3. A 2% C 5% D d b B 1000 females, all wild type 1000 males, including: 786 ++p 94 +++ 114 +0+ and and and n o+ nop n+p 6+op and n++ 5. (a) (b) Su+ host at permissive temperature Su- host at restrictive temperature 6. (a) (b) (c) (d) Either the sequence is acb, or the sequence is abc, with c very close to b. Do the same cross, but select for b+ c+ and observe distribution of a alleles. Either the sequence is bac, or it is abc, with a very close to b. b equidistant between a and c. 7. (a) a and b close together, d farther away. Approximately same distance from a to d as from b to d. 8. (a) wrong; (b) move D to between E & G; B 9. (c) 2 x 4; (d) between D & G H III C G E II I D F Eco RI fragments indicated on the outside. Sal I fragments indicated on the inside. A 10. |-------------8.5-------------|-----------4.8------------|-----------6.2----------|-------------12.2------------|---X B E B X E X B X E B X _______________________________________________________________________________________ ------3.3--------------------------------10.0-------------------------7.1---------------------------------------------6.9----------------------------------9.6-------------------------------------------