Chapter21 Model Organisms

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Chapter21 Model Organisms

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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.

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Some Important Model Organisms

• Escherichia coli and its phage (the T phage and
  phage ?)
• Bakers yeast Saccharomyces cerevisiae
• The nematode Caenorhabditis elegans
• The fruit fly Drosophila melanogaster
• The house mouse Mus musculus

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Bacteriophage
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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.

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• 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

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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

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• 2. Temperate phage
• eg. Phage ?
• Lysogeny (????)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.

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The lysogenic cycle of a bacteriophage
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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.

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

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The single-step growth curve
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• 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.

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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.

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• 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.

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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

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

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Bacteria

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

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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).

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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.

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Bacteria Exchange DNA by

• Sexual Conjugation
• Phage-Mediated Transduction
• DNA-Mediated Transformation

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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)

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• 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.

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• 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

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• 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.

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• 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 cant undergo
  genetic exchange.

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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

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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.

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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.

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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.

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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

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eg2. Gene and operon fusions created by
transopsons
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• 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.

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BAKERS YEAST, Saccharomyces cerevisiae

• Unicellular eukaryotes offer many advantages as
  experimental model systems. And the best studied
  unicellular eukaryote is the budding yeast S.
  cerevisiae.

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

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• 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.

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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.

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• 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.

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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.

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

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S. cerevisiae cells change shape as they grow

• As S. cerevisiae cells progress through the cell
  cycle. They undergo characteristic changes in
  shape.

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• 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.

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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.

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• Suitable characteristics
• Rapid generation time
• Hermaphrodite(?????) reproduction producing large
  numbers of self-progeny

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• 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.

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Figure 21-14 b The body plan of the wrom
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• 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.

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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.
• 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.

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

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THE FRUIT FLY, Drosophila melanogaster

• 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.

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• 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).

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The growth of the imaginal disks arising from
invaginations of epidermis in mid-stage embryos.
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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.

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• 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).

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• 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.

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• 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.

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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.

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• 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
• 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. one-male)

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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.

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Figure 21-20 FLP-FRT
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• 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 z/z tissue in different regions of
  an adult fly.

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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.

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• 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.

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P-elements can be used as vectors in the
transformation of the fly embryos
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• 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.

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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.

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The predominance of the mouse model

• 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.

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• 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)

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• 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.)

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Mouse Embryonic Development Depends on Stem Cells

• The first obvious diversification of cell types
  is at the 16-cell stage, called the morula .
• The cells in outer regions of the morula develop
  into the placenta .
• Cells in internal regions generate the inner
  cell mass (ICM) which is the prime source of
  embryonic stem cells.
• At the 64-cell stage the mouse embryo, called a
  blastocyst , is ready for implantation.
  Interactions between the blastocyst and uterine
  wall lead to the formation of the plancenta.

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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 of a
  female mouse.
• Third, the injected DNA integrates at random
  positions in the genome.

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• Germline transformation the offspring of
  transgenic mice also contain the foreign
  recombinant DNA.

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• 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.

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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

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• Figure 21-26 Gene knockout via homologous
  recombination

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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.

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Figure 21-27 Imprinting in the mouse
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• 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.