What is the purpose of kanamycin in this experiment?

1. In order to determine the overall chromosome map for E. coli, three Hfr strains (# 1, 2, & 3)

were obtained from F+ ancestors. The start times of transfer d

1. What is the purpose of kanamycin in this experiment?

uring conjugation matings with Fstrains carrying auxotrophic, resistance, and fermentation markers were determined for each Hfr

strain, as listed.

1. Using the times for transfer of each

maker, draw a unified chromosome map of

1. coli, showing the order and relative

distances between these markers.

1. Place the origin of transfer (site of F

insertion) on the map for each Hfr strain.

1. What is the total length of the E. coli

chromosome, in transfer minutes?

Time (min) of marker

transfer

Strain: 1 2 3

Gene

Leu 8 – 12

Ala 12 – 8

Gal 24 – –

Erm 32 4 –

Ade 36 8 –

His 50 22 44

Trp – 34 32

Ser – 40 26

2. A prototrophic Hfr strain is used to map four genes involved in the synthesis of various amino

acids in an interrupted mating experiment. A prototrophic Hfr strain with genotype leu+

val+ phe+

tyr+

kans was mated to an auxotrophic F- strain with the genotype leuval- phetyrkanr

. (No

map order is implied in the listing of the alleles and kanr

is resistance to the antibiotic

kanamycin). The cross is initiated at time=0 and at various times the mating mixture is plated

on four types of medium. Each plate contains minimal medium (MM) and kanamycin plus

specific supplements as indicated in the table. The results for each time interval are shown as

the number of colonies growing on each plate.

Suppl. Added to MM + kanamycin Time of Mating Interruption

12 min 20 min 30 min 37 min 43 min 60 min

Plate#1 leu val phe 0 0 0 2 31 72

Plate#2 val phe tyr 0 0 1 21 45 98

Plate#3 leu phe tyr 0 0 0 0 3 56

Plate#4 leu val tyr 2 19 43 67 89 134

1. What is the purpose of kanamycin in this experiment?
2. What is the order of these genes? (Write the order of transfer from first to last in left to right

order).

1. What is the distance between phe and tyr?
2. In the recipient strain, where is the ideal location of the kanr gene?
3. Bacteriophage T2 normally infects the different E. coli strains, B and C. However, certain

mutations, designated r, block growth on strain B. Ten r mutations were mapped by two-point

recombination frequencies to a small region of the T2 chromosome, shown below.

a g d j h b e f i k c

———-|—|–|————|—|———-|————————-|——|————-|——-|———-|—

0 0.1 0.2 0.3 0.4 0.5 0.6 map units

1. When the same ten r mutants were tested for complementation by mixed-infection of strain

B, certain pairs could kill the B host (+), while other pairs failed to grow completely (-).

Assuming the mutations a, b, and c are known to lie in the genes A, B, and C, use the

complementation data in the table below to mark gene boundaries on the genetic map.

a b c d e f g h I j k

a –

b + –

c + + –

d – + + –

e + + + + –

f + + + + – –

g – + + – + + –

h + – + + + + + –

I + + + + – – + + –

j – + + – + + – + + –

k + + – + + + + + + + –

1. Do the data reveal the presence of a fourth gene in this region?
2. If so, please show its location on the map.
3. Using the technique of interrupted mating, five Hfr strains were tested for the

sequence in which they transmitted a number of different genes to an F- strain. Each

Hfr strain was found to transmit its genes in a unique sequence, as follows (only the first

six genes transmitted were scored).

1. F O S R P Q
2. R S O F G Y
3. Y X W Q P R
4. W Q P R S O
5. F G Y X W Q
6. Place all markers in order on a single circular genetic map.
7. Show the location and orientation of the F factor in each Hfr strain.
8. For each of the Hfr strains, state which donor-gene marker should be selected in the

recipients after conjugation to obtain the highest proportion of recombinants that will be

converted to become Hfrs?

Strain 1

Strain 2

Strain 3

Strain 4

Strain 5

5. In order to determine the overall chromosome map for a new wild type strain of E. coli, two Hfr

strains were obtained by independent insertions of the F factor.

The start times of transfer of several genes were determined during a conjugation mating between each

Hfr strain and an F

–

recipient carrying six auxotrophic mutations.

Time (min) of marker transfer

Hfr Strain: 1 2 3

Gene

Ade 22 62 12

His 27 – 17

Ile 32 – 22

Lys 50 10 –

Bio – 14 –

Met – 20 –

1. Using the times taken to begin transfer of each marker, draw a unified chromosome map of the E.

coli chromosome, showing its total length in minutes of transfer and the order and relative distances

between these markers.

1. Show the position of the F factor in Hfr strain #2