8. Chromosomes, plasmids and gene transfer(2) Flashcards
How can DNA from a donor cell be introduced into a recipient cell? (3 methods)
1) Transformation (uptake of naked DNA)
2) Transduction (phage-mediated)
3) Conjugation (plasmid-mediated)
What does transduction require?
A bacteriophage
Is Dna degraded in tranformation?
the DNA may or may not be degraded
Are the donor and recipient cell different in conjugation?
No, the donor and recipient cell are different
Describe the mechanism of Transformation? (6)
1) DNA binds via protein
2) ONE strand enters using the DNA translocase; the second is degraded.
3) The internalised single strand is bound by RecA protein.
4) As RecA has more than one DNA binding site, it can hold the ssDNA and dsDNA together.
5) The RecA-ssDNA complex stretches the dsDNA to increase complementarity recognition (conformational proofreading)
6) Branch migration and homologous recombination follows.
(the transforming DNA doesn‘t need to be a plasmid (but often is))
What does a normal bacteriophage contain?
phage genes; transducing particles contain host genes as well
What happens in transduction?
Virus invades host cell - disrupts host cell DNA and takes one part with it when it leaves. When it invades another cells - it leaves the DNA from the initial cell. Transduction is limited by the physical capacity of the PHAGE.
What happens in conjugation?
Donor and receiving cell - parts of chromsome is transfered from cell to cell. Conjugation is least limited as its limiting factor is how long the cells are in contact for - dictates how much DNA can be transferred.
Describe Fred Griffeth’s transformation experiments?
Can you get heat inactivated bacteeria and induce immunity. Rough and smooth bacteira - capsule found in smooth bacteria. Capsule protects the cells from immune system and dehydration.
Injected heat killed bacteria into mice - mice OBVIOUSLY SURVIVED.
INjected live smooth cells - mice DIES
INjected live rough cells - mice is alive - rough cells dont have capsule.
Inject mice with heat killed smooth cells and live R cells - mice dies → live rough cells pick up DNA.
What did Oswald Avery find?
Proved that DNA was the genetic material AND the TRANSFORMING PRINCIPLE
How many proteins are required for transformation?
40+ proteins
Whys is transformation a powerful process?
Most bacteria only take up their own DNA. On chromsomes - have shorter segments of DNA which are species specific - ensures they only pick up DNA from their own species
What are the limits of transformation uptake?
1 million base pairs
What is the mechanism of transduction?
Two cycles: lytic cycle and transduction cycle.
-Phage takes part of host DNA
mechanism play large role in nature as phages are so abundant in nature .
Plaques formed by lysed bacteria following phage infection.
Does conjugation require physical contact?
U-tube experiment showing physical contact between two bacterial strains of Lederbeg and Tatum experiment was needed for genetic exchange to occur
What does mating of bacteria require?
-Mating requires pili responsible for cell paring
Some virions infect cells through pili.
Transfer of the F plasmid by conjugation
1) Pili contracts and gets cells into close proximity.
2) F plasmid gets nicked and transfered from F+ cell to F- cell.
3) Completion of DNA of complementary strand in recipient cell
4) Cells separate.
How is the F plasmid transferred?
Conjugation
How can chromosome genes be transferred ?
due to integrated F plasmid
What doe Frf stand for?
High frequency of recombination for chromosomal markers
What strain is involved in the transfer of chromosomal genes?
Hfr strain
What happens to the donated genetic material when in cell? (2)
Once in the cell, the donated genetic material can stay as a plasmid, OR it can be integrated into the host cells DNA → replicated with the host cell DN
What did availability if Hfr strains allow microbiologists to find?
Allowed microbiologists to identify the arrangement and orientation of almost all genes before the genome sequence of E. coli became known
What does longer transfer from donor to recpipient mean?
The longer the transfer from donor to recpipient takes, the further away from the integration site the marker will sit.
How long does it take to transfer F plasmid?
It takes only 5 minutes to transfer the F plasmid but much longer for part of the chromosome. Time of entry in minutes = position of the chromsome.
What is the use of transformation in the lab?
Transformation is used very widely to get plasmids or fragments of linearised DNA into numerous bacterial cell types. Some bacteria are naturally competent to be transformed, others need chemical treatment or electroporation.
What is the use of transduction in the lab?
Transduction is the second most important method and is still used to move small fragments of DNA, maybe a gene or transposon from one cell to another.
What is the use of conjugation in the lab?
Conjugation is of enormous historical interest and value but is little used these days.
