Lecture 10

Question 1

How does evolution occur?

Answer 1

Genetic diversity by recombination and mutations

Question 2

What do eukaryotes use to effect recombination?

Answer 2

Sexual cycle + meiosis

Question 3

What do prokaryotes use to effect recombination?

Answer 3

Transformation - uptake of ‘naked’ DNA
Conjugation - Utilises plasmids
Transduction - Utilises bacteriophages

Question 4

What do many bacteria, including E. coli, have as a genome?

Answer 4

Single circular dsDNA chromosome

Question 5

3 distinct types of plasmid

Answer 5

Sex plasmids
R plasmids
col plasmids

Question 6

Sex plasmids

Answer 6

F plasmid of E. coli ~100kbp - stringent replication (copy number 1-2), self mobile

Approx 35% sequences encode transfer between bacteria

Remaining sequences contain 4 insertion sequences (1 x 1S2, 2 x 1S3, 1 x 1S1000 aka gammadelta)

-Mediate transfer of bacterial genes by conjunction

• F plasmid is an episome - may exist as free circular plasmid or integrated to chromosome

Question 7

R plasmids

Answer 7

~30-100kbp

Self mobile

Encode resistance to drugs, heavy metals, toxins

Evolved a lot over last 60 years with antibiotic use

R plasmids spread through environment

Question 8

col plasmids

Answer 8

Small (mostly below <25kbp)

Encode biological factors

Relaxed replication (copy number ~30)

Don’t encode functions permitting transfer between individual bacteria

• Transfer may occur if F and R plasmids present in same cell encoding functions for contact and transfer
• Manipulated for useful vectors e.g. pGEM3zf from colE1 plasmid

Question 9

How was F plasmid mediated conjunction discovered

Answer 9

Lederberg and Tatum - 1946

• 2 auxotrophic strains mixed
• Some phototrophic colonies observed when mixture plated on minimal media
• Caused by genetic exchange
• U-tube established physical contact required

Question 10

Bacterial conjugation

Answer 10

F+ cells contacts F- cell - connection established by long F- pilus

F+ pilus contracts forming a bridge

Genetic material transferred by cytoplasmic bridge

F plasmid carries tra genes for contact and mobilisation functions - encodes pilin protein to build pilus

Question 11

F-plasmid transfer

Answer 11

• tra genes encode contact and DNA transfer functions
• Transfer initiated by introducing a nick in DNA at oriT
• 5’ end of ssDNA transferred to recipient
• Rolling circle replication forms ssDNA from F-plasmid
• DNA synthesis in F- recipient restores second strand

Question 12

Transfer of F plasmid by conjunction in E. coli

Answer 12

Transfer of F factor from donor F+ cell to recipient F- cell during F+ x F- matings

All F plasmid transferred and replicated = DNA circularised and original F- cell becomes F+ cell

Question 13

What does transfer of bacterial chromosomes require?

Answer 13

F factor becomes integrated into bacterial chromosome

Question 14

Chromosomal integration of F-plasmid

Answer 14

• Can be integrated in either orientation according to orientation of recombining sequences
• Integrated by recombination between insertion sequences of F plasmid and chromosome

-Reversible

Question 15

Hfr transfer

Answer 15

High frequency transfer

F plasmid integrated into chromosome where it encodes transfer functions

F plasmid oriT nicked

F factor initiates transfer to recipient

F plasmid transferred followed by bacterial chromosome

Transferred chromosomal DNA recombines with recipient chromosome

Question 16

Hft conjugation

Answer 16

• Conjugate with f- strains
• Unidirectional, ordered transfer of chromosomal genes from donor to recipient
• Part of F transferred first - bridge fragile and breaks
• Conjugation not species specific - Horizontal gene transfer
• DNA homology determines success of promiscuous conjugation

Question 17

Donors in mapping bacterial genes by conjugation

Answer 17

Donor: HfrH, thr+, aziR, tonR, lac+, gal+, strS

Phototrophic
Resistant to sodium azide
Infection with bacteriophage T1
Sensitive to streptomycin

Question 18

Recipients in mapping bacterial genes

Answer 18

F-, thr-, leu-, aziS, tonS, lac-, hal-, strR

Auxotrophic for threonine and leucine
Sensitive to sodium azide
Sensitive to infection by bacteriophage T1
Resistant to streptomycin

Question 19

Process of mapping bacterial genes

Answer 19

Mix bacteria

Withdraw samples at time intervals after mating commences

Break apart mating cells

Plate bacteria on selective media to determine which genes transferred from Hfr to F-

Question 19

Imprecise excision of F plasmid from bacterial chromosome

Answer 19

• F plasmid integrated into bacterial chromosome
• F factor loops out incorrectly, including a piece of the chromosome
• Single crossover generates F’lac
• F’lac+ can transfer to recipient (lac-)
• Circular DNA can be maintained without integration into chromosome
• Recipient become lac+/lac- partial diploid - Used in genetic analysis of gene regulation

Question 20

Life cycle of virulent bacterophage

Answer 20

• T4 bacteriophage uses tail fibres to stick to receptor sites on surface of E. coli
• Phage injects it’s DNA after sheath of tail constructs a hollow core through wall
• Empty capsid left as ghost, DNA hydrolysed
• Phage proteins and nucleotides produced to copy phage genome

Phage parts come together to form head, tail and tail fibres

• Phage directs production of lysozyme to hydrolyse cell wall and burst cell by osmosis, releasing new bacteriophages