Lecture 9 - bacterial conjugation

Question 1

What are 3 different means of genetic transfer?

Answer 1

Transformation
Transduction
Conjugation

Question 2

Transformation

Answer 2

Some bacteria can take up DNA from their surroundings

Question 3

Transduction

Answer 3

Incorporation of bacterial DNA into a phage for transfer

Question 4

Conjugation

Answer 4

Plasmids that copy their DNA into new cells

Question 5

Plasmids

Answer 5

Circular double stranded DNA molecules that replicate independently of the chromosome
Confer a new function upon the host cell, giving it an advantage over other bacteria

Question 6

Joshua Lederberg & Edward Tatum

Answer 6

Bacteria strain A and strain B could not grow on minimal media. A & B carry mutations that cause an inability to synthesise constituents needed for growth.
When A & B are mixed for a few hours colonies begin to grow due to the exchange of genetic material

Question 7

Minimal medium

Answer 7

The basic synthetic medium for bacterial growth without nutrient supplements

Question 8

bio-
arg-
met-

Answer 8

Required biotin/arginine/methionine added as a supplement to minimal medium

Question 9

lac-

gal-

Answer 9

Cannot use galactose/lactose as a carbon source

Question 10

Str^r

Str^s

Answer 10

Resistant to antibiotic streptomycin

Sensitive to the antibiotic streptomycin

Question 11

Bernard Davis

Answer 11

Created an experiment to demonstrate that physical contact between bacterial cells is needed for genetic recombination to occur.
A suspension of strain A which is unable to synthesise certain nutrients was placed in one arm of a U tube and strain B, which was unable to synthesise other metabolites was placed in the other arm. After several hours of incubation no colonies grew.
(The medium could move in either direction but the bacteria could not)

Question 12

William Hayes

Answer 12

Determined that genetic transfer occurred in one direction and that transfer is not reciprocal - one cell acts as the donor while the other is the recipient.

Question 13

F+

F-

Answer 13

F+ strains are those that carry the F factor and can create a pilus
F- strains are those that do not carry the F factor so cannot create a pilus

Question 14

F factor

Answer 14

A plasmid that can transfer itself between bacterial cells by conjugation which allows a copy of the plasmid to move from one cell to the next

Question 15

Transconjugants

Answer 15

Bacteria that have incorporated DNA from another bacterium by conjugation.

Question 16

What are the steps in bacterial conjugation?

Answer 16

F+ donor cells contain the F plasmid that allows them to make the F pilus and act as a genetic donor.
Contact between the donor and recipient as the F pilus recognises and binds receptor sites on the cell wall.
A relaxosome cleaves the DNA at the origin of transfer. The relaxosome associates with the T-DNA strand
A single F plasmid, beginning at the origin of transfer, enters the F- cell.

Question 17

Does gene transfer require direct contact between cells?

Answer 17

Yes, there must be direct contact between donor and recipient cells

Question 18

What are the steps to rolling circle replication?

Answer 18

One DNA strand is nicked at oriTand the 3’ OH is extended by DNA polymerase.
The 3’ end on the circle is lengthened while the growing point rolls around the circular template.
The 5’ end is displaced and forms a tail of single stranded DNA that extends from the circle.
The single stranded DNA is converted to double stranded DNA through the use od RNA primers.
The 5’ end will enter the recipient cell as a single strand.

Question 19

Hfr

Answer 19

High frequency of recombination
Strain that produces 1000X more recombinants than a normal F+ strain
The F plasmid is cut at oriT by an exonuclease and integrated into the host cell genome.

Question 20

How do Hfr strains form?

Answer 20

As plasmids are being transferred random breakage can occur which interrupts the transfer before the entire chromosome is transferred. This fragment may then recombine with the recipient chromosome.

Question 21

In Hfr how is the donor DNA incorporated into the recipients chromosome?

Answer 21

Homologous recombination

Question 22

In Hfr what happens to linear DNA that is not incorporated into the chromosome?

Answer 22

It is degraded

Question 23

How did Wollman and Jacob discover linkage maps could be constructed?

Answer 23

Use interrupted mating as a measure of time at which a donor allele begins to appear after mating.
e.g. if lac+ begins to enter F- cell 10 minutes after azi^r then these genes are 10 units apart