Conjugation mapping
- 1. CONJUGATION MAPPING OF E.COLI CHROMOSOMES ZULEKHA KHALIL MSc BIOCHEMISTRY FERGUSSON COLLEGE, PUNE
- 2. SOME TERMS 5 Types bacterial plasmid: 1. F plasmid-Contains ‘tra-gene’ responsible for conjugation 2. R plasmid-Carry genes for resistance to antibiotics 3. Col plasmid-Contain genes that code for ‘Colicins’; proteins that can kill other bacteria 4. Degradative plasmid-Enables digestion of unusual substance 5. Virulence plasmid-Turn the bacterium into a pathogen
- 3. Contd…. • Fertility factor: Allows genes to be transferred from one bacterium carrying the F-factor to another bacterium lacking the F-factor. • F factor is a self-replicating, circular DNA plasmid (1/40 the size of the main chromosome). • F+ Bacteria: Possess F-factor as a plasmid independent of bacterial genome. • F- cell: They do not contain F-factor. They act as recipient of F-factor. • Hfr cell: An Hfr cell is a bacterium with a conjugative plasmid(F-factor) integrated into its genomic DNA.
- 4. INTRODUCTION BACTERIAL SEXUAL PROCESSES • Eukaryotes have the processes of meiosis to reduce diploids to haploids, and fertilization to return the cells to the diploid state. • Bacterial sexual processes are not so regular. However, they serve the same aim: to mix the genes from two different organisms together. • The three bacterial sexual processes: • 1. Transformation: Naked DNA is taken up from the environment by bacterial cells. • 2. Conjugation: Direct transfer of DNA from one bacterial cell to another. • 3. Transduction: Use of a bacteriophage (bacterial virus) to transfer DNA between cells.
- 5. TRANSFORMATION • Very important for recombinant DNA work. • The essence of recombinant DNA technology is to remove DNA from cells, manipulate it in the test tube, then put it back into living cells. In most cases this is done by transformation. • In the case of E. coli, cells are made “competent” to be transformed by treatment with calcium ions and heat shock. E. coli cells in this condition readily pick up DNA from their surroundings and incorporate it into their genomes.
- 6. CONJUGATION • Meaning: the formation or existence of a link between things. • The temporary union of two bacteria or unicellular organisms for the exchange of genetic material. • Was first discovered by Joshua Lederberg & Edward Tatum. • William Hayes (1953) demonstrated that genetic exchange in E. coli occurs in only one direction. • Genetic transfer is mediated by sex factor F. • F is a self-replicating, circular DNA plasmid (1/40 the size of the main chromosome). • The ability to conjugate is conferred by the F plasmid.
- 7. Contd….. • F+ cells grow special tubes called “sex pilli” from their bodies. When an F+ cell bumps into an F- cell, the sex pilli holds them together, and a copy of the F plasmid is transferred from the F+ to the F-. Now both cells are F+. • F plasmid contains an origin sequence (O), which initiates DNA transfer. It also contains genes for hair-like cell surface (F-pili or sex-pili), which aid in contact between cells.
- 8. MECHANISM Figs. A and B Transfer of the F factor
- 9. Contd….. • No conjugation can occur between cells of the same mating type. • Conjugation begins when the F-plasmid is nicked at the origin, and a single strand is transferred using the rolling circle mechanism. • When transfer is complete, both cells are F + double-stranded.
- 10. RELAXASE AND RELAXOSOME • When conjugation is initiated, via a mating signal, a relaxase enzyme (an endonuclease) creates a nick in one plasmid DNA strand at the Origin of transfer, or oriT. • The relaxase may work alone or in a complex of over a dozen proteins, known collectively as a relaxosome. • The transferred, or T-strand, is unwound from the duplex plasmid and transferred into the recipient bacterium in a 5'-terminus to 3'-terminus direction. • The remaining strand is replicated, either independent of conjugative action or in concern with conjugation.
- 11. MECHANISM Contd… Each F+bacterium contains F pili Binding of F pili to specific outer membrane protein Intercellular cytoplasmic bridge formation Transfer of single strand from donor to recipient Transferred strand is converted into double stranded circle in recipient. The copy is retained in donor The newly synthesized double stranded DNA is called exogenote and native DNA as endogenote.
- 12. CONJUGATION OF Hfr CELLS • A high-frequency recombination cell (Hfr cell) (also called an Hfr strain) is a bacterium with a conjugative plasmid(often the F-factor) integrated into its genomic DNA. • No chromosomal DNA is transferred by standard sex factor F. • Transfer of chromosome DNA is facilitated by special strains of F + integrated into the bacterial chromosome by crossing over.
- 13. Contd…… • Hfr strains = high frequency recombination strains. • Discovered by William Hayes and Luca Cavalli-Sforza. • Hfr strains replicate F -factor as part of their main chromosome. • Conjugation in Hfr strains begins when F + is nicked at the origin, and F+ and bacteria chromosomal DNA are transferred using the rolling circle mechanism
- 14. MECHANISM Fig. Transfer of the Hfr F+ factor
- 15. CHARACTERISTICS • DNA transfer requires cell-cell contact. • DNA transfer occurs via a conjugal pore. • DNA transfer occurs in one direction - from donor to recipient not vice versa • DNA transfer does not require protein synthesis in donor. • DNA transfer requires energy in donor cell - primarily ATP.
- 16. CONCLUSION • The genetic information transferred is often beneficial to the recipient cell. Benefits may include antibiotic resistance, other xenobiotic tolerance, or the ability to utilize a new metabolite. Such beneficial plasmids may be considered bacterial endosymbionts. • Some conjugative elements may also be viewed as genetic parasites on the bacterium, and conjugation as a mechanism was evolved by the mobile element to spread itself into new hosts
- 17. REFERENCES http://en.wikipedia.org/ www.ncbi.nlm.nih.gov www.youtube.com AND GOOGLE
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