Intro to Plasmids Flashcards
(13 cards)
What is a plasmid?
Small, circular dsDNA that naturally exist in bacterial cells and occur in some eukaryotes.
Genes carried in plasmids provide bacteria genetic advantages e.g. antibiotic resistance
Range from 1,000bp to many
When bacterium divides, each daughter cell receives a copy of each plasmid
Bacteria can also transfer plasmids through conjugation
What are the important structural points of plasmids?
1) Origin of replication (OR)
2) Multiple cloning region (MCR)
3) Inserted gene
What are the definitions of the OR?
1) Minimal cis-acting region that can support autonomous replication of plasmid
Region where DNA strands are melted to initiate replication process; OR
3) Base(s) which leading-strand synthesis starts, replication origins contain sites that are required for interactions of plasmid-encoded and/or host-encoded proteins
What are the 3 stages to plasmid replication?
1) Initiation
2) Elongation
3) Termination
What does initiation in plasmid replication consist of?
The intro of a specific single strand break (nicking) in replication origin by Rep Protein, encoded by plasmid
Catalysed most frequently by 1/few plasmid-encoded initiation proteins that recognise plasmid-specific DNA sequences and determine point from which replication starts
What carries out elongation in plasmid replication?
DNA polymerase III holoenzyme
DNA polymerase I can also be involved at early stages with participation of other host proteins
What is a replisome?
A large protein complex that facilitates DNA replication starting at the replication origin
Mediates several enzymatic activities such as helicase, primase + DNA polymerase
Creates a replication fork to duplicate leading and lagging strand
What does the termination stage do in plasmid replication?
The nicking and rejoining of displaced ss at the site where initial single-strand break was introduced
What molecules are involved with control of replication and plasmid duplication?
1) RNA (antisense RNA)
2) DNA sequences (iterons)
3) Antisense RNA and proteins acting in concert
What are restriction enzymes (RE, also called Restriction Endonucleases)?
Proteins that cleave DNA at specific sequence sites, producing discrete fragments of DNA
Are though to protect bacterial cells from phage infection
Bacterial cells that contain REs can ‘cut up’ invasive viral DNA without damaging their own DNA
What are the 2 types of nuclease enzymes?
1) Endonucleases - cleaves phosphodiester bond within DNA
2) Exonucleases - cleave phosphodiester at terminal points on DNA
What do bacterial endonucleases rely on?
Recognition sequences to cut DNA
These can exist within either plasmids/chromosome (within chromosome, the sequence is methylated)
What happens if a bacteriophage is able to infect a host bacterial cell?
1) Bacterial endonucleases will destroy/inactivate invading DNA - preventing bacteriophage replication cycle; OR
2) The viral (phage) nucleases will bind to host DNA and destroy it
Bacteria cells can evolve a defence mechanism - methylating DNA to prevent phage enzyme binding
