Microbiology and Molecular Genetics – 502 Problem Set for L2 factor in Hfr, F' and F - strains. 1. Describe the state of the F factor 2. Why can generalized transduction transfer a large set of genes, while specialized transduction can only transfer a small set? 3. Four E. coli Hfr strains, that were all derived from the same F + strain, donate the following markers to an F - strain in the order shown: #1 Q W D M T #2 A X P T M #3 B N C A X #4 B Q W D M What is the relative position of these markers on the chromosome? What is the position and direction of each of the Hfrs? 4. In an Hfr X F - cross, leu+ enters the cell first, followed by the other markers. The Hfr strain is prototrophic and the F - strain, strain, is auxotrophic for all of the markers. What is the order of the markers in which the leu+ recombinants selected are 27% ile+, 13% mal+, 82% thr+ and 1% trp+? 5. A cross is made between an Hfr strain that is met + thi+ pur+ and an F- strain that is met - thi- pur-. Interrupted mating experiments show that the met + markers enters last. Recombinants are selected on plates supplemented with only pur and thi requirements. These cells are then tested for thi+ and pur+ and the following numbers are obtained: met +, thi+, pur+ met +, thi+, pur-
280 0
b) What is the gene order?
met +, thi-, pur+ met +, thi-, pur-
6
c) What is the map distances in recombination
52
a) Why was methionine left out of the media?
units?
6. Four E. coli Hfr strains that were all derived from the same F + strain donate the following markers in the order shown:
#1 M Z X W C #2 L A N C W #3 A L B R U #4 Z M U R B What is the order of these markers on the chromosome? What is the position and direction of each of the Hfrs? 7. Four E. coli strains (#1-4) of the genotype a+ b- are mated in all pairwise combinations with four other strains (#5-8) that have the genotype of a- b+. Cells with the a+ b+ phenotype are selected and counted. The results are shown below with M = many recombinants, L = low number of recombinants and 0 = no recombinants.
1 2 3 4 5 0 M M 0 6 0 M M 0 7 L 0 0 M 8 0 L L 0 Assign the sex type (F’, Hfr, F-) of each strain . 8. Jacob selected 8 (L1-L8) closely linked lac- mutants and attempted to order the mutants with respect to outside markers pro and ade by performing a pair of reciprocal crosses with each pair of lac mutants and selecting on media with lactose as the only carbon source. Determine the order of the mutants from the table below Cross A Hfr pro- lac-x ade+ X F- pro+ lac-y adeCross B Hfr pro- lac-y ade+ X F- pro+ lac-x adex 1 1 1 1 1 1
y 2 3 4 5 6 7
XA 173 156 46 30 168 37
XB 27 34 218 197 32 215
x 1 2 2 3 4 5
y 8 3 8 6 5 7
XA 226 24 153 20 205 199
XB 40 187 17 175 17 34
9. A particular Hfr strain normally transmits the pro+ marker last during conjugation. However in a cross with an F - strain, some pro+ recombinants are recovered early in the
mating. When these pro+ cells are mixed with an F - strain, the majority of the cells are converted to pro+ cells that carry the F factor. Explain this result. 10. A cross was made between an Hfr strain that is arg+, bio+, leu+ and an F - strain that is arg-, bio-, leu-. Interrupted mating experiments show that the arg+ markers enters last.
Arg+ cells were selected and then tested for bio+ and leu+. The following numbers were obtained: 320 arg+, bio+ leu+ a) What is the gene order? arg+, bio+, leuarg+, bio-, leu+
8
arg+, bio-, leu-
48
0
b) What is the map distances in recombination units?
Microbiology and Molecular Genetics - Problem Set #2 Answers 1. Hfr - The F factor is integrated into the chromosome. F' - The F factor is an episomal element that also contains copies of the chromosomal genes. F- - There are no F factor genes in this strain. 2. Generalized transduction occurs by packaging random fragments of the bacterial genome into the phage particle. Any fragment in the genome may be packaged. Specialized transduction occurs when the a phage lysogen is not properly excised and a region adjacent to the point of integration is included in the excised phage. Only genes that are adjacent to the point of integration will be packaged. 3. The map of these markers on the chromosome is: Hfr#1 is between Q and B in the clockwise direction Hfr#2 is between C and A in the counter-clockwise direction Hfr#3 is between Q and B in the counter-clockwisedirection Hfr#4 is between B and N in the clockwise direction 4. leu, thi, ile, mal, trp 5. a. To select for cells that have obtained the met + marker in mating. b. and c.
met
pur 52/338 15.3 m.u.
thi
6/338 1.7 m.u.
6. Hfr#1 is between U and M in the clockwise direction Hfr#2 is between L and B in the counter-clockwise direction Hfr#3 is between N and A in the clockwisedirection Hfr#4 is between Z and X in the counter-clockwise direction
7. A 0 indicates the strains are either both F - or are either Hfr or F +. An M indicates a mating between and Hfr and and F - strain. An L indciates a mating between and F + and
a F- strain. a. Place the strains in mating type groups that can not mate with each other. (2, 3, 7) vs. (1, 4, 5, 6, 8). b. Since 7 can either mate in pairs to produce L or M number of recombinants, then that indicates that it is an F - strain and therefore so is 2 and 3. c. Since 4, 5, and 6 give M recombinants when crossed with the F - strains (2,3,7), then these must be Hfr strains.
d. Since 1 and 8 give L number of recombinants when mated with F - strains (2,3,7) then these must be F + strains.
1-F+, 2-F-, 3- F-, 4-Hfr, 5-Hfr, 6-Hfr, 7-F-, 8-F+ 8. If cross A>B then the order of the markers is pro-x-y-ade. If B>A then the order of the markers is pro-y-x-ade. pro 4
5
7
1
6
3
2
8
ade
9. The Hfr has excised from the chromosome and become an F' that is carrying the pro+ gene. 10.
