Genetics: Genetics Lecture 17, 10/8 - Plasmids, Transformation, Bacteriophage

• F Factor is a plasmid. Plasmid is a small circular, extrachromosomal, piece of DNA. Plasmids are replicated when the host cell goes through DNA replication.
• Types of Plasmids

o F plasmid: contains genes necessary for a fertile cell. Allows the formation of the sexpilus. During the transfer F plasmid is transmitted to the recipient cell.
o R plasmid: two important regions, transfer region → allows the plasmid to be passed between cells, AND R-determinant → codes for resistance to an antibiotic.
o Colplasmid: commonly found in e. coli. It produces a toxin called colicin. A cell that lacks the colplasmid will be killed by the toxin. A cell WITH the plasmid will be immune to the toxin that it produces.

• Transformation (6:25) – small pieces of DNA that are transferred from one cell to the next. Two STEP Process: entry (DNA enters into the cell) AND recombination (DNA enters the host cells genome).

o Entry: requires a bacterial cell to be in a state of competence. A competent cell is one that has the ability to take up exogenous DNA. DNA enters and one of the two strands degrades.
o DNA strand looks for regions of homology, places where it is similar to the host cells DNA. It will then Bind to the region of homology.
o Recombination: host genomes DNA is removed and the DNA that is entered becomes part of the host genome.
o Following replication you have one cell with two copies of your newly replicated DNA.

o 1954 – first demonstration that bacterial genes can be linked by transformation (in pnuemococcus).
o When transformation occurs 19,000 to 20,000 base pairs of DNA are brought it. This is a small gragment in comparision to a large genome.
o If two genes are co-transformed (transformed on the same piece of DNA) they are relatively close to each other.
o EX. Mutation on B. subtilis

• Have a mutation that leaves a cell that requires galactose to grow.
• Take a number of pieces of DNA from the B. subtilis genome and transform them into the cell. (20:40)
• We will transform the mutant strain and determine what percentage of cells NO LONGER require galactose. We will use the strains from 4 separate quadrants along the genomes. Whatever piece it is nearest will have a greater percentage of cells that will be able to grow without galactose (the mutant trait). This in turn will allow you to have a general idea of where the mutation is.
• The higher the percentage of co-transformation between two genes, the more closely linked in the genome they are.

o Transduction mapping – bacteriaphage is used to transmit DNA from one cell to the next. The ADVANTAGE to transduction mapping is that we can take up more DNA.
o T4 phage : You have DNA surrounded by the phage head. The phage head is an Icosohedral (20 sided) protein coat. Head is connected to the tail. In the Tail there is a contractile sheath. The contractile sheath functions to punch open holes in the host cell membrane. Tail fibers recognize and bind the host cell specifically

o Bind to the host cell, mediated by the tail fibers.
o The contractile sheath opens a hole in the membrane of the cell and the DNA is injected.
o Viral DNA hijacks the replication and transcription and translation machinery of the cell. It then starts to replicate itself
o Start to assemble mature phage.
o You eventually reach a critical mass. When 200 phages have been produced the phages produce a compound called lysozyme. This functions to lyse the host cell and release the new phages.

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