User:Kalearose.fajardo/Origin of transfer

Old article: https://en.wikipedia.org/w/index.php?title=Origin_of_transfer&oldid=947288549

Lead
The origin of transfer (oriT) is a non-coding DNA sequence of up to 500 base pairs long on a plasmid that is necessary so that bacterial conjugation can begin. Conjugation is the process by which a host bacterial cell takes up a plasmid containing target DNA, which often contains recombinant genes. The first step of this process occurs when the relaxosome protein complex recognizes the oriT sequence.

History
Joshua Lederberg and Edward Tatum were the first to show in 1946 that E. coli bacterium possess a method of gene transfer through a sexual process, known as bacterial conjugation. The first oriT to be identified and cloned was on the RK2 (IncP) conjugative plasmid, which was done by Guiney and Helinski in 1979.

Features
oriT sequences can be as short as 40 bp or as long as 500 bp. The sequences vary between conjugative and nonconjugative plasmids as well as within these groups. This is a cis-acting DNA region, meaning that it acts on the DNA and is transferred with it. Within the oriT sequence, there is a nicn nicking region that is about 10 bp long. This is where the relaxosome first binds. There is also a termination sequence upstream of the nicn site.

Reaction Mechanism
A plasmid's oriT sequence serves as both a recognition point and a substrate for the enzymes in the relaxosome. The first step of bacterial conjugation occurs at the nicn site of the oriT region of the plasmid. Relaxase enzymes, otherwise known as DNA strand transferases that are part of the relaxosome complex, catalyze a strand- and site-specific phosphodiester bond cleavage at the nicn site.

Every plasmid's DNA only binds with a specific relaxase. This reaction is a trans-esterification, which produces a nicked double-stranded DNA with the 5' end bound to a tyrosine residue in the relaxase. The relaxase then moves toward the 3' end of the strand to unwind the DNA in the plasmid.

The other strand of the plasmid, the strand that was not nicked by the relaxase, is a template for further synthesis by DNA polymerase.

Once the relaxase reaches the upstream section of the oriT again where there is an inverted repeat, the process is terminated by reuniting the ends of the plasmid and releasing a single-stranded plasmid in the recipient.

Genetic engineering
Main article: Bacterial conjugation

Conjugation allows for the transfer of target genes to many recipients, including yeast, mammalian cells, and diatoms.

Diatoms could be very useful plasmid hosts as they have the potential to autotrophically produce biofuels and other chemicals. There are some methods for genetic transfer for diatoms, but they are slow compared to bacterial conjugation. By designing plasmids for the diatoms P. tricornutum and T. pseudonana based on sequences for yeast and developing a method for conjugation from E. coli to the diatoms, researchers hope to advance genetic manipulation in diatoms.

One of the main problems in using bacterial conjugation in genetic engineering is that certain selectable markers on the plasmids generate bacteria that have resistance to antibiotics like ampicillin and kanamycin.