24 BIO 326R Conjugation - BIO 326R Conjugation Conjugation...

Info iconThis preview shows pages 1–8. Sign up to view the full content.

View Full Document Right Arrow Icon
BIO 326R Conjugation
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Conjugation Transfer of DNA from one bacteria to another No phage intermediate, no cell death, no “free” DNA intermediate Unidirectional Cell-cell contact required
Background image of page 2
History: Lederberg and Tatum Two auxotrophic strain Strains have different mutations / phenotype / genotype Mix (or don’t, as a control) in nutrient broth to allow cell contact and growth Plate on minimal media Simultaneous complementation of 3 mutations – virtually no chance of simple reversion accounting for this bio- phe- cys- thr+ leu+ thi+ bio+ phe+ cys+ thr- leu- thi- Growth only occurs when strains have been mixed. Phenotype of cells is bio+ phe+ cys+ thr+ leu+ thi+ Mix Incubate in rich media (hours) Plate on minimal media
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
History: Bernard Davis  Some explanations for the previous data: A transducing phage Some bacteria die and DNA is exchanged Soluble metabolic intermediates are diffusing between the bacteria (they’re still auxotrophs) Add a filter to the incubation in a “U tube” set up Subsequent plating on minimal media showed no growth – no wt bacteria produced as long as filter was in place Control with no filter = growth Demonstrates necessity for cell – cell contact bio- phe- cys- thr+ leu+ thi+ bio+ phe+ cys+ thr- leu- thi- filter
Background image of page 4
Conjugation  Pili (typically encoded on plasmid) project from surface of one bacteria and make contact with another bacteria Pili retracts, putting bacteria in close physical contact Genetic material passed from donor to recipient Donor also retains a copy F+ cell (has conjugal pili) F- cell (no conjugal pili)
Background image of page 5

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
F factor 60-100 kb plasmid F stands for fertility Contains ~20+ tra genes, spread over ~30kb of sequence. These encode proteins necessary for transfer of DNA from one cell to another DNA nicking and unwinding Pili structural genes oriT: origin of transfer – site (a specific sequence) where plasmid gets nicked to initiate transfer of DNA Bacteria harboring F factor may be called F+, hfr or F’ It’s a little confusing, as F+.hfr and F’ are all “F positive,” meaning they contain F DNA. Cells without F DNA are “F negative” or “F minus.”
Background image of page 6
Transfer Relaxosome: complex of proteins which bind to oriT and cause a single stranded nick TraI: Bifunctional - nuclease and helicase activities TraY and IHF (Integration Host Factor - a host chromosomal gene. Not derived from plasmid) both bind DNA sequences near oriT nick site TraY and IHF binding facilitate binding of TraI and formation of the relaxosome, leading to nicking of one strand Nicking is both site and strand specific oriT TraI TraY IHF Y Y Y TraI TraI
Background image of page 7

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 8
This is the end of the preview. Sign up to access the rest of the document.

Page1 / 18

24 BIO 326R Conjugation - BIO 326R Conjugation Conjugation...

This preview shows document pages 1 - 8. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online