Chapter21 Model Organisms

1. Chapter21Model Organisms 叶行 200431060007 生物学基地班

2. Model Organism • Two important feature of all model systems • first, the availability of powerful tools of and study the organism genetically. Second, ideas, methods, tools, and strains could be shared among scientists investigating the same organism, facilitating rapid progress.

3. Some Important Model Organisms • Escherichia coli and its phage (the T phage and phage λ) • Baker’s yeastSaccharomyces cerevisiae • The nematodeCaenorhabditis elegans • The fruit flyDrosophila melanogaster • The house mouseMus musculus

4. Bacteriophage

5. Bacteriophage • Bacteriophage (and viruses in general) offer the simplest system to examine the basic processes of life. Phage typically consist of a genome (DNA and RNA, most commonly the former) packaged in a coat of protein subunits, some of which form a head structure (in which the genome is stored) and some a tail stricture.

6. 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. • Phage come in two basic types-lytic and temperate. The former, examples of which include the T phage, grow only lytically

7. 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

9. 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.

10. The lysogenic cycle of a bacteriophage

11. Assays of phage growth • For bacteriophage to be use ful as an experimental system, methods are needed to propagate and quantify phage. To quantify the numbers of phage particles in a solution, a plaque assay is used.

12. Plaques firmed by phage infection of a lawn of bacterial cells.

13. Plaque is the result of multiple round of infection, a circular clearing in the otherwise opaque lawn of densely grown uninfected bacterial cells. Knowing the number of plaques on a given plate, and the extent to which the original stock was diluted before plating, makes it trivial to calculate the number of phage in that original stock.

14. The single-step growth curve

15. The time lapse between infection and release of progeny is called the latent period, and the number of phage released is called the burst size.

16. Phage crosses an complementation tests • Differences in host range and plaque morphologies of the phage were very often the result of genetic differences between otherwise identical phage. • The ability to perform mixed infection-in which a single cell is infected with two phage particles at once-makes genetic analysis possible in two ways.

17. First, it allow one to perform phage crosses. • Second, co-infection also allow one to assign mutations to complementation groups; that is, one can identify when two or more mutations are in the sane or in different genes.

18. Transduction and recombinant DNA • The process involves a site-specific recombination event, and if that event occurs at slightly the wrong position, phage DNA is lost and bacterial DNA included is as known as specialized transduction

19. Because of the ability to promote specialized transduction, it was natural that phage λ was chosen as one of the original cloning vectors. • Many different λ vectors were developed, all differing in the restriction sites used and in how recombinant phage could be identified

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

21. 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).

23. 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.

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

25. 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)

26. 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.

27. 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

28. 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.

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

30. 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

32. 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”.

33. 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.

34. Transposons Can Be Used to Generate Insertional Mutations and Gene and Operon Fusions 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.

36. 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

37. eg2. Gene and operon fusions created by transopsons

38. Large quantities of bacterial cells can be grown in a defined and homogenous physiological state. • It is easier to purify protein complexes harboring precisely engineered alterations or to overproduce and obtain individual proteins in large quantities. • It is much simpler to carry out DNA replication, gene transcription, protein synthesis, etc. in bacteria than in higher cells.

39. BAKER’S YEAST, Saccharomyces cerevisiae • Unicellular eukaryotes offer many advantages as experimental model systems. And the best studied unicellular eukaryote is the budding yeast S. cerevisiae.

40. Figure 21-10 The lifecycle of the budding yeast S. cerevisiae

41. These cell types can be manipulate to perform a variety of genetic assays. • Genetic complementation can be performed the two mutations whose complementation is being tested. • If the mutations complement each other, the diploid will be a wild type for mntations can be made in haploid cells in which there is only a single copy of that gene.

42. Generating precise mutations in yeast is easy • The genetic analysis of S. cerevisiae is further enhanced by the availability of techniques used to precisely and rapidly modify individual genes.

44. The ability to make such precise changes in the genome allows very detailed questions concerning the function of particular genes or their regulatory sequences to be pursued with relative ease.

45. S. cerevisiae has a small, well-characterized genome • Because of its rich history of genetic studies and its relatively small genome, S. cerevisiae was chosen as the first eukaryotic ( nonviral ) organism to have its genome entirely sequenced. This landmark was accomplished in 1996.

46. The availability of the complete genome sequence of S. cerevisiae has allowed “genome-wide” approaches to studies of this organisn.

47. S. cerevisiae cells change shape as they grow • As S. cerevisiae cells progress through the cell cycle. They undergo characteristic changes in shape.

49. Simple microscopic observation of S. cerevisiae cell shape can provide a lit of information about the events occurring inside the cell. • A cell that lacks a bud has yet to start replicating its genome. A cell with a very large bud is almost always in the process of executing chromosome segregation.

50. caenorhabditis elegans • In 1965 Sydney Brenner settled on the small nematode worm caenorhabditis elegans to study the important questions of development and the molecular basis of behavior, because it contained a variety of suitable characteristics. • And due to its simplicity and experimental accessibility, it is now one of the most completely understood metazoan.