Host restriction and modification

Question 1

Restriction enzymes

Answer 1

Can be used to restrict viral DNA infection.
Bacteria has immunity to it from methylation of its own DNA.
They are endonucleases that cut specific sequences in DNA.

Question 2

Type II restriction enzymes

Answer 2

Recognize palindromic sequences, generate staggered cuts, allows easier recombination. This restriction enzyme is mostly used exclusively for cloning.

Question 3

Type III

Answer 3

bind to specific sites but cut DNA 24 to 36 bp

Question 4

Methylase

Answer 4

Prevents cleaving of restriction enzymes.

There are more methylase enzyme than restriction enzymes produced.

Question 5

Bacteriophage lambda

Answer 5

Attach to bacterium cell surface.
DNA injected through both membrane
DNA is linear with sticky ends
Ligase converts linear DNA into circular DNA. The site of which is annealed is called the cos-site.
DNA polymerase replicated the DNA, it effectively replicates 2 DNA strands.

Question 6

Terminase

Answer 6

Recognises a cos site in the DNA, it cleaves a staggered break at the cos site by the terminase. DNA is translocated into the newly synthesised phage head.
The signal to terminate the process is when the phage head comes into contact with another cos site.

Tail protein is then added (r protein), this tail can penetrate the peptidoglycan layer.

a cycle takes about 35 minutes.

Question 7

Gene library

Answer 7

1) Cloning vector is required.
Lambda replacement vectors - capacity 20kb
A viral DNA is made of Head, Tail, Stuffer region, Regulatory genes.
The stuffer region is cleaved and an equivalent length of DNA is replaced.

Question 8

in vitro packaging

Answer 8

2 Strains of lysogens
First strain has a mutation, so no head protein is produced
Second strain has a mutation that does not encode for protein D
Hence, they cannot undergo lysis

Both cell extracts can be combined, which can be added to the DNA concatamers. This generates new DNA particles.

Question 9

Cosmids - capacity 40kb

Answer 9

A plasmid which contains a lambda cos site.
clone cos DNA into a plasmid
Cos plasmid is cleaved by BgiII to linearise it
It is joined with genomic DNA
The sticky ends is complimentary to the Genomic DNA hence a long strand if formed.

Question 10

BACs - capacity 300kb

Answer 10

Bacterial artificial chromosome, derived from F plasmid.

Question 11

Phage biology

Answer 11

T4 can mediate generalised transduction