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Welcome Each of You to My Molecular Biology Class.

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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 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 Model 1: BACTERIOPHAGE CHAPTER 15 The Genetic Code 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 1.Lytic phage: eg. T phage infect a bacterial cell DNA replication coat proteins expression host cell lysed to release the new phage

14 Figure 21-1 The lytic growth cycle

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 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 Bacteriophage Figure 21-4 Latent period- the time lapse between infection and release of progeny. Burst size-the number of phage released

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 Bacteriophage  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. eg. Phage λ

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

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

34 Assays of Bacteria Growth Bacteria  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).

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

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: Bacteria  Sexual Conjugation  Phage-Mediated Transduction  DNA-Mediated Transformation

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, called promiscuous 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 Bacteria  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.

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.

51 eg2. Gene and operon fusions created by transopsons Promoter-less lacZ Reporter gene Gene fusion: a fusion in which the reporter is joined both transcriptionally and translationally to the target gene. Figure 21-9

52 Studies on the Molecular Biology of Bacteria Have Been Enhanced by Recombinant DNA Technology, Whole- Genome Sequencing, and Transcriptional Profiling Bacteria

53 Biochemical Analysis Is Especially Powerful in Simple Cells with Well- Developed Tools of Traditional and Molecular Genetics Bacteria

54  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.

55 Bacteria Are Accessible to Cytological Analysis Bacteria Despite their simplicity and the absence of membrane-bound cellular compartments, bacteria are accessible to the tools of cytology, such as: Immunofluorescence microscopy Fluorescence microscopy Fluorescence in situ hybridization (FISH)

56 Phage and Bacteria Told Us Most of the Fundamental Things about the Gene Bacteria There are countless examples where fundamental processes of life were understood by choosing these simplest of systems.

57 Model 3: BAKER ’ S YEAST Saccharomyces cerevisiae Model 3: BAKER ’ S YEAST Saccharomyces cerevisiae CHAPTER 15 The Genetic Code 5/08/05

58 Features of S. cerevisiae  Have small genomes  Can be grown rapidly in the lab  Central characteristics: they contain a discrete ( 不连续的 ) nucleus with multiple linear chromosomes packaged into chromatin; their cytoplasm includes a full spectrum of intracellular organelles and cytoskeletal structures.

59 The Existence of Haploid and Diploid Cells Facilitate Genetic Analysis of S. cerevisiae Saccharomyces cerevisiae S. Cerevisiae can grow in either a haploid state (one copy of each chromosome) or diploid state (two copies of each chromosome). Conversion between the two states is mediated by mating (haploid to diploid) and sporulation (diploid to haploid).

60 Figure S. Cerevisiae exist in three forms: two haploid cell types, a and а, and the diploid product of mating between these two.

61 Application in the Lab  Genetic complementation: by mating two haploid strains, each contains one of the two mutations whose complementation is being tested.  Testing the function of an individual gene: mutations can be made in haploid cells in which there is only a single copy of that gene.

62 Generating Precise Mutations in Yeast Is Easy Saccharomyces cerevisiae When linear DNA with ends homologous to any given region of the genome is introduced into S. cerevisiae cells, very high rates of homologous recombination are observed resulting in the transformation.

63 Figure Recombinational transformation in yeast This property can be used to make precise changes within the genome, allowing very detailed questions to be elucidated.

64 S. Cerevisiae Has a Small, Well-Characterized Genome S. Cerevisiae was the first eukaryotic organism to have its genome entirely sequenced. (1996) 1.3X10 6 bp, approximately 6,000 genes. The availability of the complete genome sequence has allowed “genome-wide” approaches to studies of this organism. Saccharomyces cerevisiae

65 S. Cerevisiae Cells Change Shape as They Grow Saccharomyces cerevisiae  S. Cerevisiae divides by budding. The bud grows until it reaches a size approximately equal to the size of the mother cell and is released from it.  The emergence of a new bud is tightly connected to the initiation of DNA replication.

66 Start replicating its genome Chromosome segregation Figure The mitotic cell cycle in yeast

67 Model 4 : THE NEMATODE WORM, Caenorhabditis elegans CHAPTER 15 The Genetic Code 4/22/05

68 Caenorhabditis elegans  Suitable characteristics: Rapid generation time Hermaphrodite( 雌雄同体的 ) reproduction producing large numbers of “ self- progeny ”

69 Sexual reproduction so that genetic stocks could be constructed A small number of transparent cells so that development could be followed directly

70 C.elegans Has a Very Raplid Life Cycle  C.elegans is cultured on petri dishes, fed a simple diet of bacteria and grow well at 25°C. Caenorhabditis elegans

71 Figure The lifecycle of the C. elegans 12 h Eggs Juvenile Adult Death 12h 40h 15d

72  Dauer Forming under stressful condition Resistant to environmental stresses Living many months while waiting for environmental conditions to improve

73 C. elegans Is Composed of Relatively Few, Well Studied Cell Lineages Caenorhabditis elegans Figure a

74 Figure b The body plan of the wrom gonad: 生殖腺 oocyte: 卵母细胞 uterus: 子宫 vulva: 阴孔 pharynx: 咽 intestine: 肠 anus: 肛门

75  Mutations that disrupt the formation of the vulva form a “ bag of worms ” (the hatched worms devour their mother and become trapped inside her skin).

76  The genes are components of a highly conserved receptor tyrosine kinase signaling pathway that controls cell proliferation.

77 Mutations that inactivate this pathway eliminate vulva development. Mutations that activate this pathway cause overproliferation of the vulva precursor cells.

78 The cell Death Pathway Was Discovered in C. elegans  Cell death is under genetic control (a mutated ced gene).  Analysis of the ced mutants showed that the cell commits suicide. In males, a cell known as the linker cell is killed by its neighbor. Caenorhabditis elegans

79 RNAi Was Discovered in C. elegans  RNAi silencing Caenorhabditis elegans Enzyme Dicer makes siRNAs siRNAs direct a complex called RISC to repress gene in three ways Translational inhibition Motifying promoters Digesting mRNA

80 Figure 17-30

81  In 1998, RNAi was discovered in C. elegans, which is significant in two respects: RNAi appears to be universal. Experimental investigation reveals the molecular mechanisms.

82 Model 5: THE FRUIT FLY, Drosophila melanogaster CHAPTER 15 The Genetic Code 4/22/05

83 In 1908, Thomas Hunt Morgan and his research associates at Columbia University placed rotting fruit on the window ledge of their laboratory. Among the menagerie of creatures that were captured, the fruit fly emerged as the animal of choice.

84 Drosophila Has a Rapid Life Cycle Drosophila melanogaster Figure The rapid life cycle of Drosophila

85  The growth of the imaginal disks: arising from invaginations of epidermis in mid-stage embryos. Figure Imaginal disks in Drosophila

86 There is disks for appendages, eyes, antennae, the mouthparts, and genitalia. Disks are composed of fewer than 100 cells in the embryo but thousands of cells in mature larvae.

87 The wing imaginal disk has become an important model system for the control of complex patterning processes by gradients of secreted signaling molecules.

88 The First Genome Maps Were Produced in Drosophila  Useful features of the flies in experimental research : Fecundity Rapid life cycle Four chromosomes (two large autosomes, a smaller X, and a very small fourth chromosome) Polytene chromosomes Drosophila melanogaster

89 Endoreplication in the absence of mitosis generates enlarged chromosomes in some tissues of the fly Figure Polytene chromosomes

90  Two major discoverise by the Morgan lab in 1910 : Mendel ’ s first law: Genes are located on chromosomes, and each gene is composed of two alleles that assort independently during meiosis. Mendel ’ s second law: Genes located on separate chromosomes segregate independently

91  By the 1930s, extensive genetic maps were produced that identified the relative positions of numerous genes. (the distances between linked genes mapped by recombination frequencies)

92  Large-scale “ genetic screens ” are performed by feeding adult males a mutagen which cause mutations, and then mating them with normal females.(A variety of method used to study these mutations)

93  Method one Bridges used polytene chormosomes to determine a physical map of the Drosophila genome. Bridges identified 5000 “ bands ” on the four chromosomes and established a correlation between the bands and the locations of genetic loci.

94 For example Female fruit flies that contain the white mutation and a small deletion in the other X chromosome, which removes polytene bands 3C2-3C3, exhibit white eyes. This type of analysis led to the conclusion that the white gene is located between polytene bands 3C2-3C3 on the X chromosome

95  Method two Balancer chromosomes contain inversions Figure 21-16

96 Such balancers fail to undergo recombination with the native chromosome. Thus, it is possible to maintain fruit flies that contain recessive, lethal mutations.

97 Genetic Mosaics Permit the Analysis of Lethal Genes in Adult Files  Mosaics are animals that contain small patches of mutant tissue in a generally “ normal ” genetic background.  The most spectacular genetic mosaics are gyandromorphs. Drosophila melanogaster

98 Figure Gyandromorphs

99 Rarely, one of the two X chromosomes is lost at the first mitotic division. Sexual identity in flies is determined by the number of X chromosomes. (two- female; one-male)

100 Suppose that one of the X chromosomes contains the recessive white allele. Then one half of the fly, the male half, has white eyes. While the other female half, has red eyes.

101 The yeast FLP Recombinase Permits the Efficient Production of Genetic Mosaics  The frequency of mitotic recombination was greatly enhanced by the use of the FLP recombinase from yeast.  FLP recognizes FRT and catalyzes DNA rearrangement. Drosophila melanogaster

102  FRT sequences were inserted near the centromere of each of the four chromosomes using P-element transformation.  Heterozygous flies contain a null allele in gene Z on one chromoso- me and a wild-type copy of that gene on the other.

103  In transgenic strains that contain FLP protein coding sequence under the control of heat-inducible hsp70 promoter, FLP is synthesized upon heat shock.

104  FLP binds to the FRT motifs in the two homologs containing gene Z and catalyze mitotic recombination. Figure FLP-FRT

105 It Is Easy to Create Transgenic Fruit Flies that Carry Foreign DNA  P-elements are transposable DNA that can cause hybrid dysgenesis ( 杂交不育 ).(how? ) Drosophila melanogaster

106 Figure hybrid dysgenesis The F1 progeny are often sterile, when mating females from the “ M ” strain with males from the “ P ” strain.

107 P-elements encode both a repressor of transposition and a transposase that promotes mobilization. The repressor is expressed in the developing P eggs. Thus M eggs lack the repressor that inhibits p-element mobilization.

108 Sometimes the P-elements insert into genes that are essential for the development of progenitors of the gametes ( 配偶子 ), and, as a result, the adult flies derived from the these matings are sterile.

109  P-elements can be used as vectors in the transformation of the fly embryos. Figure 21-22

110 A full length P-element transposon is 3 kb in length. It contains inverted repeats at the termini that are essential for excision and insertion.

111 Recombinant DNA is inserted into defective P-elements that lack the internal genes encoding repressor and transposase. Transposase is injected along with the recombination P- element vector.

112 The recombinant P-elements insert into random positions in the pole cells. The amount of recombination p- element and transposase is calibrated so that, on average, a given pole cell receives just a single integrated p-element.

113 The embryos are allowed to develop into adults and then mated with tester strains. The recombinant P-element contains a “ marker ” gene such as white +.

114 P-element transformation is routinely used to identify regulatory sequences. It can also be used to examine protein coding genes in various genetic backgrounds.

115 Model 6: THE HOUSE MOUSE, Mus musculus CHAPTER 15 The Genetic Code 4/22/05

116  The mouse is an excellent model for human development and disease, although, the life cycle of the mouse is slow by the standard of the nematode worm and fruit fly. The predominance of the mouse model

117 The mouse provides the link between the basic principles, discovered in simpler creatures like worms and flies, and human disease. The chromosome complement is similar between the mouse and human (autosomomes and X,Y sex chromosomes)

118 Extended regions of a given mouse chromosome contain “ homologous ” regions of the corresponding human chromosomes. (more than 85% of the mouse genes are correspond to human genes.)

119 Mouse Embryonic Development Depends on Stem Cells  The first obvious diversification of cell types is at the 16-cell stage, called the morula ( 桑椹胚 ). The cells in outer regions of the morula develop into the placenta ( 胎盘 ). Cells in internal regions generate the inner cell mass (ICM) which is the prime source of embryonic stem cells. Mus musculus

120  At the 64-cell stage the mouse embryo, called a blastocyst ( 胚泡 ), is ready for implantation. Interactions between the blastocyst and uterine wall lead to the formation of the plancenta.

121  Then the embryo enters gastrulat- ion ( 原肠胚 ), and the ICM forms all three germ layers: endoderm ( 内胚 层 ), mesoderm ( 中胚层 ), ectoderm ( 外胚层 ). The first stage in gastrulation is the subdivision of the ICM into two cell lays: an inner hypoblast ( 内胚层 ) and an outer epiblast ( 外胚层 ).

122 Then a groove called primitive streak ( 原条 ) forms along the length of the epiblast and the cells that migrate into the groove form the internal mesoderm. The anterior end of the primitive streak is the node.

123  Shortly thereafter, a fetus emerges that contains a brain, a spinal cord, and internal organs (eg: heart and liver).

124 Figure overview of mouse embryogenesis

125 It is Easy to Introduce Foreign DNA into the Mouse Embryo  Create transgenic mice by microinjection method. First, Inject DNA into the egg pronucleus. Second, place the embryos into the oviduct ( 输卵管 ) of a female mouse. Third, the injected DNA integrates at random positions in the genome. Mus musculus

126 Figure 21-24

127  Germline transformation: the offspring of transgenic mice also contain the foreign recombinant DNA.

128 Figure  A transgenic strain of mice was created that contains a portion of the Hoxb-2 regulatory region attached to a lacZ report gene. There are two bands of staining detected in the hindbrain region of 10.5 day embryos.

129 Homologous Recombination Permits theSelective Ablation of Individual Genes 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. Mus musculus

130  Gene disruption experiments They are done with embryonic stem (ES) cells. A recombinant DNA is created that contains a mutant form of the gene of interest. The method is used to create a cell line lacking any given gene.

131 The process of the experiment First, designing the recombination vector. It contains the modified target gene, the NEO gene (downstream of the target gene), the region of homology with the host cell chromosome (downstream of and flanking NEO) and a marker (TK, gene for thymidine kinase).

132 Second, transform the vector into ES cells. Third, select for NEO. Only the cells which undergo double recombination with the host cell chromosome can survive in the neomycin containing medium.

133 Fourth, select against TK. If illicit recombination occurs, TK gene will frequently be contained. In this case, the cells which undergo illicit recombination will die in the GANC containing medium.

134 Fifth, harvest the homologous recombination ES cells and inject them into the ICM of normal blastocysts. Sixth, insert the hybrid embryo into the oviduct of a host mouse and allowed to develop to term.

135 Figure 21-26

136 Mice Exhibit Epigenetic Inheritance  Parental imprinting: only one of the two alleles for certain genes is active, because the other copy of is selectively inactivated either in the developing sperm cell or the developing egg. Mus musculus

137 Figure imprinting in the mouse See the detail in chapter 17

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