1. Chapter 21 Model Organism 2003 级生科 2 班 苏亮 200331060158

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.  The choice of a model organism depends on what question is being asked.  In this chapter we will describe some of the most commonly studied experimental organisms and advantages of each as a model system.

4. 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.

5.  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.

6.  FIGURE 21-1 The lytic growth cycle of bacteriophage

7.  Temperate phage can also replicate lytically. But they can adopt an alternative developmental pathway called lysogeny. (figure 21-2)  In this integrated, repressed state the phage is called a prophage.  The lysogenic state can be maintained in this way for many generations but is also poised to switch to lytic growth at any time. This switch from the lysogenic to lytic pathway, called induction.

8.  FIGURE 21-2

9. 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.

10.  Figure 21-3 Plaques firmed by phage infection of a lawn of bacterial cells.

11.  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.

12. The single-step growth curve  This classic experiment revealed the life cycle of a typical lytic phage and paved the way for many subsequent experiments that examined that life cycle in detail. The essential feature of this procedure is the synchronous infection of a population of bacteria and the elimination of any re- infection by the progeny.

13.  Figure 21-4 The single-step growth curve

14.  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.

15. 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.

16.  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.

17. 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.

18.  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.

19. Bacteria  The attraction of bacteria such as E. coli or B. subtilis as experimental systems is that they are relatively simple cells and can be grown and manipulated with comparative ease.  Molecular biology owes its origin to experiments with bacterial and phage model systems.

20. Assays of bacterial growth  Bacterial cells are large enough （ about 2µm in length ） to scatter light, allowing the growth of a bacterial culture to be monitored conveniently in liquid culture by the increase in optical density.

21.  Figure 21-5 bacterial growth curve

22.  The number of bacteria can be determined by diluting the culture and plating the cells o solid (agar) medium in a petri dish. 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.

23. Bacteria exchange DAN by sexual conjugation, phage-mediated transduction, and DAN-mediated transformation  Bacterial often harbor autonomously replicating DNA elements known as plasmids.

24.  Figure 21-6 the three forms of F-plasmid carry cells

25.  The F-factor can undergo conjugation only with other E.coli strains, promiscuous conjugative plasmids provide a convenient means for introducing DNA into bacterial strains that are otherwise lacking in their own systems of genetic exchange.

26.  Yet another powerful tool for genetic exchange is phage-mediated transduction.  Generalized transduction is mediated by phage that occasionally package fragment of chromosomal DNA during maturation of the virus rather than viral DNA.

27.  Figure 21-7 Phage-mediated generalized transduction

28.  Another kind of phage-mediated transduction is called specialized transduction. It involves a lysogenic phage such as λ that has incorporated a segment of chromosomal DNA in place of a segment of phage DNA.

29.  DNA-mediated transformation: certain experimentally important bacterial species possess a natural system of genetic exchange that enables them to take up and incorporate linear, naked DNA into their own chromosome by recombination.  Often the cells must be in a specialized state known as “genetic competence” to take up and incorporate DNA from their environment.

30. Bacterial plasmids can be used as cloning vectors  Circular DNA elements in bacterial known as plasmids can serve as convenient vectors for bacterial DNA as well as foreign DNA.

31. Transposons can be used to generate insertional mutations and gene and operon fusion  Transposons are enormously useful tools for carrying out molecular genetic manipulations in bacterial.

32.  Figure 21-8 Transposon-generated insertional mutagenesis

33.  It have two important advantages over traditional mutations induced by chemical mutagenesis.  One is that the insertion of a transposon into a gene is more lifely to result in complete inactivation of the gene than a simple nucleotide switch created by a mutagen.  The second is that,having inactivated the gene, the presence of the inserted DNA makes it easy to isolate and clone that gene.

34.  Transposons can also be used to create gene and operon fusions on a genome- wide basis. such a fusion is know as an operon or transcriptional fusion.

35.  Figure 21-9 Transposon-generated lacZ fusion

36.  And a fusion in which the reporter is joined both transcriptionally and translationally to the target gene is known as a gene fusion

37. Studies on the molecular biology of bacteria have been enhanced by recombinant DNA technology, whole-genome sequencing, and transcriptional profiling

38.  With the advent of recombinant DNA technologies revolutionized molecular biological studies of higher cells. But they have had an impact on the study of bacterial model systems as well.  The availability of microarrays representing all of the genes in a bacterium has made it possible to study gene expression on a genome basis.

39.  And the availability of whole-genome sequences and promiscuous conjugative plasmids has created opportunities for carrying out molecular genetic manipulations in bacterial species that otherwise lack sophisticated, traditional tools of genetics.

40. Biochemical analysis is especially powerful in simple cells with well- developed tools of traditional and molecular genetics  There are three reasons for this:

41.  first, large quantities of bacterial cells can be grown in a defined and homogenous physiological state.  Second, the tools of traditional and molecular genetics.  Third, the machinery for carrying out DNA replication, gene transcription, protein synthesis, and so forth is much simpler (having far fewer components) in bacterial than in higher cells.

42. Bacterial are accessible to cytological analysis  Despite their small size, bacteria are accessible to the tools of cytology, such as immunofluoresence microscopy for localizing proteins in fixed cells with specific antibodies, fluorescence microscopy with the Green Fluorescent Protein for localizing proteins in living cells, and fluorescence in situ hybridization (FISH) for localizing chromosomal regions and plasmids within cells.

43.  Cytological methods are an important part of the arsenal for molecular studies on the bacterial cell.

44. Phage and bacteria told us most of the fundamental things about the gene  Molecular biology owes its origin to experiments with bacterial and phage model systems. Indeed, groundbreaking work with a pneumococcus bacterium led to the discovery that the genetic material is DNA. Since then, experiments with E.coli and its phage have the way.

45.  There are countless examples where, by choosing these simplest of systems, fundamental processes of life were understood. An important example comes from the classic work of Seymor Benzer, who examined intensely a single genetic locus in phage T4, called r Ⅱ.

46. 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.

47. The existence of haploid and diploid cells facilitate genetic analysis of S. cerevisiae  S. cerevisiae exists in three forms. Two haploid cell types, a and α, and the diploid product of mating between these two.

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

49.  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.

50. 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.

51.  Figure 21-11 recombinational transformation in yeast

52.  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.

53. 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.

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

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

56.  Figure 21-12 The mitotic cell cycle in yeast

57.  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.

58. THE NEMATODE WORM, 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.

59. C. elegans has a very rapid life cycle  At 25 ℃ fertilized embryos of C. elegans complete development in 12 hours and hatch into free-living animals capable of complex behaviors.  The first stage juvenile(L1) passes through four juvenile stages(L1-L4) over the course of 40 hours to become a sexually mature adult.

60.  Figure 21-13 The life cycle of the worm, C.elegans

61.  Under stressful conditions, the L1 stage animal can enter an alternative developmental stage in which it forms what is called a dauer.  Dauers are resistant to environmental stresses and can live many months while waiting for environmental conditions to imptove.

62. C. elegans is composed of relatively few, well studied cell lineages  C. elegans has a simple body plan. Its lineages is relatively few and well studied.

63.  Figure 21-14 The body plan of the worm

64.  Among genes that function to control the generation, specification, and differentiation of the vulva cells are components of a highly conserves receptor tyrosine kinase signaling pathway that controls cell proliferation.  Many of the mammalian homologs of these genes are oncogenes and tumor- supressorgenes that when altered canlead to cancer. But in C. elegans, mutations that inactivate this pathway eliminate vulva development.

65. The cell death pathway was discovered in C. elegans  The most notable achievement to date in C. elegans research has been the elucidation of the molecular pathway that regulates apoptosis or cell death.  Analysis of the ced mutants showed that, in all but one case, developmentally programmed cell death is cell autonomous, that is, the cell commits suicide.

66.  Cell death is as important as cell proliferation in development and disease and is the focus of intense research to develop therapeutics for the control of cancer and neurodegenerative diseases.

67. RNAi was discovered in C. elegans  In 1998 a remarkable discovery was announced. The introduction of dsRNA into C. elegans silenced the gene homologous to the dsRNA. It significant in two respects.

68. One is that RNAi appears to be universal since introduction of dsRNA into nearly all animal, fungal, or plant cells leads to homology-directed mRNA degradation.  The second was the rapidity with which experimental investigation of this mysterious process revealed the molecular mechanisms.

69. THE FRUIT FLY, Drosophila melanogaster Drosophila has a raid life cycle  The salient features of the Drosophila life cycle are a very rapid period of embryogenesis, followed by three period of larval growth prior to metamorphosis.

70.  Figure 21-15 The Drosophila life cycle

71.  One of the key processes that occurs during larval development is the growth of the imaginal disks, which arise from invaginations of the epidermis in mid-stage embryos.  Imaginal disks differentiate into their appropriate adult structures during metamorphosis (or putation).

72.  Figure 21-16 Imaginal disks in Drosophila

73. The first genome maps were produced in Drosophila  Morgan labs studied on Drosophila in 1910 led to two major discoveries: genes are located on chromosomes, and each gene is composed of two alleles that assort independently during meiosis; genes located on separate chromosomes segregate independently, whereas those linked on the same chromosome do not.

74.  Hermann J. Muller provided the first evidence that environmental factors can cause chromosome rearrangements and genetic mutations.  Bridges used the polytene chromosomes to determine a physical map of the Drosophila genome (the first produced for any organism).

75.  Figure 21-17 Genetic maps, polytene chromosome, and deficiency mapping

76.  A variety of additional genetic methods were create to establish the fruit fly as the premiere model organism for studies in animal inheritance.  For example, balancer chromosomes were created that contain a series of inversions relative to the organization of the native chromosome.

77.  Figure 21-18 Balancer chromosome

78.  Embryos that contain two copies of the balancer chromosome die because some of the inversions produce recessive disruptions in critical genes.  In addition, embryos that contain two copies of the normal chromosome die because they are homozygous for the eve null mutation.

79. Genetic mosaics permit the analysis of lethal genes in adult flies  Mosaics are animals that contain small patches of mutant tissue in a generally “normal” genetic background.  The analysis of genetic mosaics provided the first evidence that Engrailed is required for subdividing the appendages and segments of flies into anterior and posterior compartments.  The most spectacular genetic mosaics are gynandromorphs.

80.  Figure 21-19 Gyandromorphs

81.  These are flies that are literally half male female. The X instability occurs only at the first division.  And the “line” separating the male and female tissues is random. Its exact position depends on the orientation of the two daughter nuclei after the first cleavage.

82. The yeast FLP recombinase permits the efficient production of genetic mosaics  Drosophila possesses several favorable attributes for molecular studies and whole- genome analysis. Most notably, the genome is relatively small.  The frequency of mitotic recombination was greatly enhanced by the use of the FLP recombinase from yeast.

83.  Figure 21-20 FLP-FRT

84.  This method is quite efficient. In fact, short pulse of heat shock are often sufficient to produce enough FLP recombinase to produce large patches of zˉ/zˉ tissue in different regions of an adult fly.

85. It is easy to create transgenic fruit flies that carry foreign DNA  P-elements are transposable DNA segments that are the causal agent of a genetic phenomenon called hybrid dysgenesis.

86.  Figure 21-21 Hybrid dysgenesis

87.  P-element excision and insertion is limited to the pole cells, the progenitors of the gametes (sperm in males and eggs in females).  P-elements are used as transformation vectors to introduce recombinant DNAs into otherwise normal strains of flies.

88.  Figure 21-22 P-element transformation

89.  This method of P-element transformation is routinely uses to identify regulatory sequences such as those governing eve stripe 2 expression.  In addition, this strategy is used to examine protein coding genes in various genetic backgrounds.

90. The House Mouse, Mus musculus  The mouse enjoys a special status due to its exalted position on the evolutionary tree: it is a mammal and, therefore, related to humans.  The mouse provides the link between the basic principles, discovered in simpler creatures like worms and flies, and human disease.

91. Mouse Embryonic Development Depends on Stem Cells  Their small size prohibits grafting experiments of the sort done in zebrafish and frogs, but microinjection methods have been developed for introducing.  Figure 21-23 shows an overview of mouse embryogenesis.

92.  Figure 21-23 Overview of mouse embryogenesis

93. It Is Easy to Introduce Foreign DNA into the Mouse Embryo  DNA is injected into the egg pronucleus, and the embryos are places into the oviduct of a female mouse and allowed to implant and develop.  The injected DNA integrates at random positions in the genome

94.  Figure 21-24 Creation of transgenic mice by microinjection of DNA into the egg pronucleus

95.  Figure 21-25 In situ expression patterns of embryos obtained from transgenic mice

96. Homologous Recombination Permits the Selective Ablation of Individual Genes  The single most powerful method of mouse transgenesis is the ability to disrupt, or “knock out ， ” single genetic loci. This permits the creation of mouse models for human disease.  Gene disruption experiments are done with embryonic stem (ES) cells

97.  Figure 21-26 Gene knockout via homologous recombination

98. Mice Exhibit Epigenetic Inheritance  Studies on manipulated mouse embryos led to the discovery of a very peculiar mechanism of non-Mendelian, or epigenetic, inheritance.  This phenomenon is known as parental imprinting.

99.  Figure 21-27 Imprinting in the mouse

100.  The basic idea is that only one of the two alleles for certain genes is active.  It has been suggested that imprinting has evolved to protect the mother from her own fetus.