Welcome Each of You to My Molecular Biology Class

1. Welcome Each of You to My Molecular Biology Class

2. Molecular Biology of the Gene, 5/E--- Watson et al. (2004) Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods 3/22/05

3. Part V: Methods Ch 20: Techniques of Molecular Biology Ch 21: Model Organisms

4. CHAPTER 21 Model Organisms • Molecular Biology Course

5. Model Organisms • Fundamental problems are solved in the simplest and most accessible system in which the problem can be addressed. • These organisms are called model organisms.

6. Some Important Model Organisms • Escherichia coli and its phage (the T phage and phage λ) • Baker’s yeast Saccharomyces cerevisiae • The nematode Caenorhabditis elegans • The fruit fly Drosophila melanogaster • The house mouse Mus musculus

7. Features of Model Systems • The availability of powerful tools of traditional and molecular genetics. • The study of each model system attracted a critical mass of investigators. (Ideas,methods, tools and strains could be shared)

8. HOW to choose a model organism? It depends on what question is being asked. When studying fundamental issues of molecular biology, simpler unicellular organisms or viruses are convenient. For developmental questions, more complicated organisms should be used.

9. CHAPTER 15 The Genetic Code Model 1: BACTERIOPHAGE 5/08/05

10. Bacteriophage (Viruses) • The simplest system • Their genomes are replicated only after being injected into a host cell. • The genomes can recombine during these infections.

11. Figure Bacteriophage

12. Each phage attaches to a specific cell surface molecule (usually a protein) and so only cells bearing that “receptor” can be infected by a given phage.

13. Two Basic Types • Lytic phage: eg. T phage infect a bacterial cell DNA replication coat proteins expression host cell lysed to release the new phage

14. The lytic growth cycle Figure 21-1

15. 2. Temperate phage: eg. Phage λ Lysogeny (溶源途径)—the phage genome integrated into the bacterial genome and replicated passively as part of the host chromosome, coat protein genes not expressed. The phage is called a prophage. Daughter cells are lysogens.

16. Figure 21-2 The lysogenic cycle of a bacteriophage

17. The lysogenic state can switch to lytic growth, called induction. Excision of the prophage DNA DNA replication Coat proteins expression Lytic growth

18. Figure 16-24 Growth and induction of λ lysogen

19. Assays of Phage Growth • Progagate phage: by growth on a suitable bacterial host in liquid culture. • Quantify phage: plaque (嗜菌斑) assay Bacteriophage

20. Progagate phage • Find a suitable host cell that supports the growth of the virus. • The mixture of viruses and bacteria are filtered through a bacterial-proof filter.

21. Quantify phage • Phage are mixed with and adsorb to bacterial cells. • Dilute the mix. • Add dilutions to “soft agar” (contain many uninfected bacterial cells). • Poured onto a hard agar base. • Incubated to allow bacterial growth and phage infection.

22. Soft agar Hard agar a petri dish

23. This circle-of-death produces a hole or PLAQUE in a lawn of living cells. These plaques can be easily seen and counted so that the numbers of virus can be quantitated. As the viruses replicate and are released, they spread and infect the nearby cells.

24. The Single-Step Growth Curve Latentperiod-the time lapse between infection and release of progeny. Burstsize-the number of phage released Bacteriophage Figure 21-4

25. The Single-Step Growth Curve • It reveals the life cycle of a typical lytic phage. • It reveals the length of time it takes a phage to undergo one round of lytic growth, and also the number of progeny phage produced per infected cell.

26. Method 1. Phage were mixed with bacterial cells for 10 minutes. (Long enough for adsorption but too short for further infection progress.) 2. The mixture is diluted by 10,000. (Only those cells that bound phage in the initial incubation will contribute to the infected population; progeny phage produced from those infections will not find host cells to infect.)

27. 3. Incubate the dilution. At intervals, a sample can be removed from the mixture and the number of free phage counted using a plaque assay.

28. Phage Crosses and Complementation Tests Bacteriophage Mixed infection: a single cell is infected with two phage particles at once.

29. Mixed infection (co-infection) 1. It allows one to perform phage crosses. If two different mutants of the same phage co-infect a cell, recombination can occur between the genomes. The frequency of this genetic exchange can be used to order genes on the genome.

30. 2. It allows one to assign mutations to complementation groups. If two different mutant phage co-infect the same cell and as a result each provides the function that the other was lacking, the two mutations must be in different genes (complementation groups). If not, the two mutations are likely located in the same gene.

31. Transduction and Recombinant DNA • During infection, a phage might pick up a piece of bacterial DNA (mostly happens when a prophage excises form the bacterial chromosome). • The resulting recombinant phage can transfer the bacterial DNA from one host to another, known as specialized transduction. Bacteriophage eg. Phage λ

32. CHAPTER 15 The Genetic Code Model 2: BACTERIA 5/08/05

33. Features of bacteria • a single chromosome • a short generation time • convenient to study genetically

34. Assays of Bacteria Growth • Bacteria can be grow in liquid or on solid (agar) medium. • Bacterial cells are large enough to scatter light, allowing the growth of a bacterial culture to be monitored in liquid culture by the increase in optical density (OD). Bacteria

35. Bacterial cells can grow exponentially when not over-crowded, called exponential phase. Figure 21-5 Bacteria growth curve • As the population increase to high numbers of cells, the growth rate slows, called stationary phase.

36. Quantify bacteria • Dilute the culture. • Plate the cells on solid medium in a petri dish. • Single cells grow into colonies; count the colonies. • Knowing how many colonies are on the plate and how much the culture was diluted makes it possible to calculate the concentration of cells in the original culture.

37. Bacteria Exchange DNA by: • Sexual Conjugation • Phage-Mediated Transduction • DNA-Mediated Transformation Bacteria

38. We use genetic change to: • Map mutations. • Construct strains with multiple mutations. • Build partially diploid strains for distinguishing recessive from dominant mutations and for carrying out cis-trans analyses.

39. Sexual Conjugation Plasmids: autonomously replicating DNA elements in bacteria. Some plasmids are capable of transferring themselves from one cell to another. eg. F-factor (fertility plasmid of E.coli)

40. F+ cell: cell harboring an F-factor. • Hfr strain: a strain harboring an integrated F-factor in its chromosome. • F’-lac: an F-factor containing the lactose operon. Figure 21-6

41. F’ plasmid is a fertility plasmid that contains a small segment of chromosomal DNA. • F’-factors can be used to create partially diploid strains. • eg. F’-lac

42. F-factor-mediated conjugation is a replicative process. The products of conjugating are two F+ cells. • The F-factor can undergo conjugation only with other E.coli strains.

43. Some plasmids can transfer DNA to a wide variety of unrelated strains, calledpromiscuous conjugative plasmids. • They provide a convenient means for introducing DNA into bacteria strains that can’t undergo genetic exchange.

44. Phage-mediated transduction • Generalized transduction: A fragment of chromosomal DNA is packaged instead of phage DNA. When such a phage infects a cell, it introduces the segment of chromosomal DNA to the new cell. • Specialized transduction

45. Figure 21-7 Phage-mediated generalized transduction

46. DNA-mediated transformation • Some bacterial species can take up and incorporate linear, naked DNA into their own chromosome by recombination. • The cells must be in a specialized state known as “genetic competence”.

47. Bacterial Plasmids Can Be Used as Cloning Vectors • Plasmid: circular DNA in bacteria that can replicate autonomously. • Plasmids can serve as vectors for bacterial DNA as well as foreign DNA. • DNA should be inserted without impairing the plasmid replication. Bacteria

48. Transposons Can Be Used to Generate Insertional Mutations and Gene and Operon Fusions Bacteria eg1. Transposons that integrate into the chromosome with low-sequence specificity can be used to generate a library of insertional mutations on a genome-wide basis.

49. Figure 21-8 Transposon-generated insertional mutagenesis

50. Insertional mutations generated by transposons have two advantages over traditional mutations. • The insertion of a transposon into a gene is more likely to result in complete inactivation of the gene. • Having inactivated the gene, the inserted DNA is easy to isolate and clone that gene.