Plasmid Biology

What is a plasmid?

A plasmid is a small cellular inclusion consisting of a ring of DNA that is not in a chromosome but is capable of autonomous replication.

Typically, plasmids have the following features:

• They are stably inherited in an extrachromosomal state, i.e. are separate from chromosomal DNA and capable of replicating independently
  
  

α-complementation

• Occurs when two inactive fragments (α-acceptor and α-donor respectively) of E. coli β-galactosidase associate to form a functional enzyme.
• The α-acceptor fragment is generated by deletion of 5’ region of the lacZ gene which encodes the initiating methionine residues. This causes translation to begin at a downstream methionine residue and this generates a carboxy-terminal fragment of the enzyme.
• On the other hand, the α-donor fragment is the amino-terminal fragment generated by deletion or chain-terminal mutations in the structural gene.

Structure of β-galactosidase

• β-galactosidase is a tetramer composed of four identical monomers of 1023 amino acids each, which are folded into five sequential domains.
• The amino-terminal segment of the monomer is called the complementation region.

DNA Ligases

Ligation → a process that involves the formation of 4 phosphodiester (PD) bonds -- 2 at each end of the molecule between neighbouring 3'-OH and 5'-PO4

DNA Ligases

• Exquisite specificity for nicked DNA
• In E. coli:
• homologous and monomeric
• polypeptide chain with molecular weight of 75 KDa
• require NAD+ as a co-factor
• In T4 phages, mammalian and plant cells:
• polypeptide chain with molecular weight of 68 KDa
• requires ATP as a co-factor and energy source

Restriction Endonucleases

Discovery

• In his 1969 paper, Werner Arber proposed that host-controlled restriction in bacteriophages occurs as result of enzymatic cleavage of infecting DNA molecules by what he termed as endonuclease while refering to endonuclease R (later to be renamed as EcoB)
• Arber, along with Struat Linn received the Nobel Prize in Physiology and Medicine in 1978 for the discovery of restriction "enzymes" or "endonucleases"
• They also proposed that DNA modification is the result of DNA methylation by DNA "methylases"
• Shortly after the discovery, another restriction enzyme was found in E. coli (EcoK)

Nomenclature

Biology of DNA Restriction

Restriction-Modification

• Phenomenon first described 40 yrs ago in viruses
• Restriction occurs by an endonucleolytic cleavage of foreign DNA
• 2 different types:
• one where only DNA that carries specific modifications is cleaved (host DNA avoids restriction because it is not modified)
• other where cellular DNA is protected from restriction by modification i.e. by methylation of adenosyl or cytosyl residues within the sequences recognized by Restriction Enzymes -- The classical R-M systems

Restriction-Modification (R-M) systems