What is genetic material? Griffith experiment 1928

1. What is genetic material? Griffith experiment 1928

3. DNA • Watson-Crick model 1953

10. DNA polymerase I and III • DNA ligases • Primase • DNA replication is semiconservative!

11. Meselson-Stahl experiment 1958

12. oriC and dnaA Boxes

13. Cis acting sites: on this side of (acting only on the DNA, they made of • Trans acting (proteins) : on the other side of (acting on any DNA)  dnaA, B, C, G,

15. Termination

16. Bacterial genetic information: • On bacterial DNA • On plasmids • On bacteriphages • On transposons

17. BActerial Genome -Usually 1 chromosome Circular or linear No histon proteins

18. In circular bacterial DNA the replication begins at the ori locus • Ends at ter locus

19. Plasmids: ds DNA; circular Various copy number 800-300 000 bp long Carry genes providing advantages for the bacterium

20. Transposons (IS seequences) Can couple their replication to the cell division • Their propagation depends on the integration with the bacterial replicon • The insertion sites are not spesific

21. Bacteriophages Viruses of the bacteria Ds/ss DNA,ds/ss RNA Lytic or temperate phages (prophage) Different propagation strategies

23. Gene transfer among bacteria • Vertical transfer • Lateral or horizontal transfer - conjugation - transduction - transformation

24. Conjugation Most frequently plasmids are transferred Tra gene products are needed F+ E. Coli Sex pilus Hfr R plasmids

27. Interrupted Mating • Chromosome transfer from the Hfr into the F- is slow: it takes about 100 minutes to transfer the entire chromosome. • The conjugation process can be interrupted using a kitchen blender. • By interrupting the mating at various times you can determine the proportion of F- cells that have received a given marker. • This technique can be used to make a map of the circular E. coli chromosome.

28. Transduction • General Phage Life Cycle 1. Phage attaches to the cell and injects its DNA. • 2. Phage DNA replicates, and is transcribed into RNA, then translated into new phage proteins. • 3. New phage particles are assembled. • 4. Cell is lysed, releasing about 200 new phage particles. • Total time = about 15 minutes.

29. Generalized Transduction • Some phages, such as phage P1, break up the bacterial chromosome into small pieces, and then package it into some phage particles instead of their own DNA. • These chromosomal pieces are quite small: about 1 1/2 minutes of the E. coli chromosome, which has a total length of 100 minutes. • A phage containing E. coli DNA can infect a fresh host, because the binding to the cell surface and injection of DNA is caused by the phage proteins. • After infection by such a phage, the cell contains an exogenote (linear DNA injected by the phage) and an endogenote (circular DNA that is the host’s chromosome). • A double crossover event puts the exogenote’s genes onto the chromosome, allowing them to be propagated.

30. Transduction Mapping • Only a small amount of chromosome, a few genes, can be transferred by transduction. The closer 2 genes are to each other, the more likely they are to be transduced by the same phage. Thus, “co-transduction frequency” is the key parameter used in mapping genes by transduction. • Transduction mapping is for fine-scale mapping only. Conjugation mapping is used for mapping the major features of the entire chromosome.

31. Specialized Transduction • Some phages can transfer only particular genes to other bacteria. • Phage lambda (λ) has this property. To understand specialized transduction, we need to examine the phage lambda life cycle. • lambda has 2 distinct phases of its life cycle. The “lytic” phase is the same as we saw with the general phage life cycle: the phage infects the cell, makes more copies of itself, then lyses the cell to release the new phage.

32. Lysogenic Phase • The “lysogenic” phase of the lambda life cycle starts the same way: the lambda phage binds to the bacterial cell and injects its DNA. Once inside the cell, the lambda DNA circularizes, then incorporates into the bacterial chromosome by a crossover, similar to the conversion of an F plasmid into an Hfr. • Once incorporated into the chromosome, the lambda DNA becomes quiescent: its genes are not expressed and it remains a passive element on the chromosome, being replicated along with the rest of the chromosome. The lambda DNA in this condition is called the “prophage”. • After many generations of the cell, conditions might get harsh. For lambda, bad conditions are signaled when DNA damage occurs. • When the lambda prophage receives the DNA damage signal, it loops out and has a crossover, removing itself from the chromosome. Then the lambda genes become active and it goes into the lytic phase, reproducing itself, then lysing the cell.

33. Transduction • Phage mediated recombination

34. Transformation Competent cells Artificially (forced) with CaCl or temperature shock

36. Genetıc Mapping • Sequencing of the gene • Cloning the gene • Gene labeled  Hybridization  Localization of the gene on the bacterial genome

37. Nucleic acid amplification • PCR and other technologies • Real time PCR