H igh F requency of R ecombination (Hfr)

2. High Frequency of Recombination(Hfr) ...bacteria exhibiting a high frequency of recombination, …the F factor is integrated into the chromosomal genome.

3. F factor and Chromosomal DNA are Transferred

4. Double Crossover Recombination Requires Crossing over

5. Incomplete Transfer of DNA • Interrupted Mating: a break in the pilus during conjugation stops the transfer of DNA, • Transfer occurs at a constant rate, • provides a means to map bacterial genes.

6. How Do You Interrupt Bacterial Mating spread on agar mate for specified time frappe

7. Hfr and Mapping HfrH strs(sensitive to streptomycin) thr+ (able to synthesize the amino acid threonine) azir(resistant to sodium azide) tonr(resistant to bacteriophage T1) lac+ (able to grow with lactose as sole source of carbon) gal+ (able to grow with galactose as sole source of carbon) F- strr(resistant to streptomycin thr- (threonine auxotroph) azis (sensitive to sodium azide) tons (sensitive to phage T1) lac- (unable to grow on lactose) gal- (unable to grow on galactose)

8. Hfr and Mapping HfrH strs(sensitive to streptomycin) thr+ (able to synthesize the amino acid threonine) F- strr(resistant to streptomycin) thr- (threonine auxotroph) Streptomycin kills the HfrH cells in the mating mix. No threonine kills the F-cells in the mating mix.

9. Hfr and Mapping HfrH azir(resistant to sodium azide) tonr(resistant to bacteriophage T1) lac+ (able to grow with lactose as sole source of carbon) gal+ (able to grow with galactose as sole source of carbon) F- azis (sensitive to sodium azide) tons (sensitive to phage T1) lac- (unable to grow on lactose) gal- (unable to grow on galactose)

10. Interrupting Bacterial Mating spread on selective media mate 9 min blend

11. Replica Plating After 9 minutes, only azide resistant cells grow.

12. 10 Minutes Azide, and bacteriophage resistant cells grow.

13. 15 Minutes Azide, and bacteriophage resistant cells, and lactose utilizing cells.

14. 18 Minutes All recombinants grow.

15. % cells with markers

16. Bacterial Map Distances units = minutes

17. HfrH

18. F factor inserts in different regions of the bacterial chromosome, Also inserts in different orientations.

19. Replication Origin Hfr Order of transfer strain H thr azi ton lac pur gal his gly thi 1 thr thi gly his gal pur lac ton azi 2 lac pur gal his gly thi thr azi ton 3 gal pur lac tonazi thr thi gly his

20. Indicates direction of transfer. A a A F factor A a Hfr F- Hfr DNA that is not incorporated in the F- strand, and DNA that has crossed out of the F- strand is digested.

21. A A Hfr F- F factor A transfers first. A A Hfr F- A transfers last. Leading Gene: the first gene transferred is determined empirically.

22. Hfr Order of transfer strain H thr azi ton lac pur gal his gly thi 1 thr thi gly his gal pur lac ton azi 2 lac pur gal his gly thi thr azi ton 3 gal pur lac tonazi thr thi gly his

24. Microbes… …in the news.

25. Hfr and Mapping HfrH strs(sensitive to streptomycin) thr+ (able to synthesize the amino acid threonine) azir(resistant to sodium azide) tonr(resistant to bacteriophage T1) lac+ (able to grow with lactose as sole source of carbon) gal+ (able to grow with galactose as sole source of carbon) F- strr(resistant to streptomycin thr- (threonine auxotroph) azis (sensitive to sodium azide) tons (sensitive to phage T1) lac- (unable to grow on lactose) gal- (unable to grow on galactose)

26. Hfr and Mapping HfrH strs(sensitive to streptomycin) thr+ (able to synthesize the amino acid threonine) F- strr(resistant to streptomycin) thr- (threonine auxotroph) Streptomycin kills the HfrH cells in the mating mix. No threonine kills the F-cells in the mating mix, * also, azide, T1 phage, and a lack of carbon source.

27. Hfr and Mapping HfrH azir(resistant to sodium azide) tonr(resistant to bacteriophage T1) lac+ (able to grow with lactose as sole source of carbon) gal+ (able to grow with galactose as sole source of carbon) F- azis (sensitive to sodium azide) tons (sensitive to phage T1) lac- (unable to grow on lactose) gal- (unable to grow on galactose)

28. Bacterial Map Distances units = minutes

29. E. coli Map • 0 minutes is at the threonine, • 100 minutes is required to transfer complete genome,

30. Typical Problem

32. combine

34. combine

35. +

36. Join Maps 11.5 minutes 26 minutes Refer to partial maps for map distances.

37. Practice • Insights and Solutions, #2, • Problem 7.17, 7.18, 7.19.

38. Transformation • heritable exchange brought about by the incorporation of exogenous DNA, • usually DNA from same, or similar species.

39. Not all cells are competent to receive DNA. Donor and Recipient

40. Competence …a transient state or condition in which a cell can bind and internalize exogenous DNA molecules, …often a result of severe conditions, • heat/cold, • starvation, etc.

41. Competent Cell Genes are expressed that produce proteins that, in turn, span the cell membrane.

42. Exogenous DNA Binds Receptor

43. ...one strand of the exogenous DNA is degraded also. Complementary Strand Degraded

44. Heteroduplex Exogenous DNA Incorporated

45. Cell Divides

46. Transformation and Mapping • transformed DNA is generally 10,000 - 20,000 base pairs in length, • carries more than one gene, • When two or more genes are received from the same transformation event, they are said to be co-transformed.

47. Linkage in Bacteria • genes that are closer together, have a higher probability of being co-transformed, • higher probability of being on same donor DNA, • lower chance of crossover event between genes, • probability of transformation by two separate events is low, • linkage in bacteria refers to proximity.

49. Transposable Elements …a segment of DNA that can move to, or move a copy of itself to another locus on the same or a different chromosome (hopping DNA), …may be a single insertion sequence, or a more complex structure (transposon) consisting of two insertion sequences and one or more intervening genes.

50. Transposon: carries one or more genes. Transposable Elementsmobile DNA