Welcome Each of You to My Molecular Biology Class

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Welcome Each of You to My Molecular Biology Class
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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
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Part V Methods
Ch 20 Techniques of Molecular Biology Ch 21
Model Organisms
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• Molecular Biology Course

• CHAPTER 21
• Model Organisms

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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.

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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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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)

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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.

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CHAPTER 15 The Genetic Code
Model 1 BACTERIOPHAGE
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Bacteriophage (Viruses)

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

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Figure Bacteriophage
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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.

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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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The lytic growth cycle
Figure 21-1
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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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Figure 21-2 The lysogenic cycle of a
bacteriophage
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• The lysogenic state can switch to lytic growth,
  called induction.
• Excision of the prophage DNA
• DNA replication
• Coat proteins expression
• Lytic growth

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Figure 16-24 Growth and induction of ? lysogen
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Assays of Phage Growth

• Progagate phage
• by growth on a suitable bacterial host in
  liquid culture.
• Quantify phage
• plaque (???) assay

Bacteriophage
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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.

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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.

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Soft agar
Hard agar
a petri dish
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As the viruses replicate and are released, they
spread and infect the nearby cells.

• 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.

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

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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.)
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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.
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Phage Crosses and Complementation Tests

Bacteriophage
Mixed infection a single cell is infected with
two phage particles at once.
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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.
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• 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.
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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 ?
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CHAPTER 15 The Genetic Code
Model 2 BACTERIA
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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).

Bacteria
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• 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.

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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

Bacteria
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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.

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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.

Figure 21-6
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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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Figure 21-7 Phage-mediated generalized
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.

Bacteria
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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.
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Figure 21-8 Transposon-generated insertional
mutagenesis
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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
Promoter-less lacZ
Reporter gene
Figure 21-9
Gene fusion a fusion in which the reporter is
joined both transcriptionally and translationally
to the target gene.
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Studies on the Molecular Biology of Bacteria Have
Been Enhanced by Recombinant DNA Technology,
Whole-Genome Sequencing, and Transcriptional
Profiling

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

Bacteria
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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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Bacteria Are Accessible to Cytological Analysis

Despite their simplicity and the absence of
membrane-bound cellular compartments, bacteria
are accessible to the tools of cytology, such as
Bacteria

• Immunofluorescence microscopy
• Fluorescence microscopy
• Fluorescence in situ hybridization (FISH)

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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.
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CHAPTER 15 The Genetic Code
Model 3 BAKERS YEAST Saccharomyces cerevisiae
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Features of S. cerevisiae

• Have small genomes
• Can be grown rapidly in the lab
• Central characteristics
• they contain a discrete (????) nucleus with
  multiple linear chromosomes packaged into
  chromatin
• their cytoplasm includes a full spectrum of
  intracellular organelles and cytoskeletal
  structures.

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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).
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S. Cerevisiae exist in three forms two haploid
cell types, a and ?, and the diploid product of
mating between these two.
Figure 21-10
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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.

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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.
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Figure 21-11 Recombinational transformation in
yeast
This property can be used to make precise changes
within the genome, allowing very detailed
questions to be elucidated.
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S. Cerevisiae Has a Small, Well-Characterized
Genome
Saccharomyces cerevisiae

• S. Cerevisiae was the first eukaryotic organism
  to have its genome entirely sequenced. (1996)
• 1.3X106 bp, approximately 6,000 genes.
• The availability of the complete genome sequence
  has allowed genome-wide approaches to studies
  of this organism.

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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.

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Start replicating its genome
Figure 21-12 The mitotic cell cycle in
yeast
Chromosome segregation
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CHAPTER 15 The Genetic Code
Model 4 THE NEMATODE WORM, Caenorhabditis
elegans
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Caenorhabditis elegans

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

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

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C.elegans Has a Very Raplid Life Cycle
Caenorhabditis elegans

• C.elegans is cultured on petri dishes, fed a
  simple diet of bacteria and grow well at 25C .

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Eggs
12h
Juvenile
40h
Adult
12 h
15d
Death
Figure 21-13 The lifecycle of the C. elegans
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• Dauer
• Forming under stressful condition
• Resistant to environmental stresses
• Living many months while waiting for
  environmental conditions to improve

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C. elegans Is Composed of Relatively Few, Well
Studied Cell Lineages
Caenorhabditis elegans
Figure 21-14 a
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Figure 21-14 b The body plan of the wrom
gonad??? oocyte???? uterus?? vulva??
pharynx? intestine? anus??
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• 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).

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

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

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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
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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
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Figure 17-30
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• 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.

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CHAPTER 15 The Genetic Code

• Model 5 THE FRUIT FLY, Drosophila melanogaster

4/22/05
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• 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.

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Drosophila Has a Rapid Life Cycle
Figure 21-15 The rapid life cycle of Drosophila
Drosophila melanogaster
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• The growth of the imaginal disks arising from
  invaginations of epidermis in mid-stage embryos.

Figure 21-16 Imaginal disks in Drosophila
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• 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.

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

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The First Genome Maps Were Produced in Drosophila
Drosophila melanogaster

• 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

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Endoreplication in the absence of mitosis
generates enlarged chromosomes in some tissues of
the fly
Figure 21-17 Polytene chromosomes
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• Two major discoverise by the Morgan lab in 1910
• Mendels first law Genes are located on
  chromosomes, and each gene is composed of two
  alleles that assort independently during meiosis.
  
• Mendels second law Genes located on separate
  chromosomes segregate independently

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• 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)

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• 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)

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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 .

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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

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Method two

• Balancer chromosomes contain inversions

Figure 21-16
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• Such balancers fail to undergo recombination with
  the native chromosome. Thus, it is possible to
  maintain fruit flies that contain recessive,
  lethal mutations.

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Genetic Mosaics Permit the Analysis of Lethal
Genes in Adult Files
Drosophila melanogaster

• 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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Figure 21-19 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 one-male)

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• 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.

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The yeast FLP Recombinase Permits the Efficient
Production of Genetic Mosaics
Drosophila melanogaster

• The frequency of mitotic recombination was
  greatly enhanced by the use of the FLP
  recombinase from yeast.
• FLP recognizes FRT and catalyzes DNA
  rearrangement.

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• 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.

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

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FLP binds to the FRT motifs in the two homologs
containing gene Z and catalyze mitotic
recombination.
Figure 21-20 FLP-FRT
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It Is Easy to Create Transgenic Fruit Flies that
Carry Foreign DNA
Drosophila melanogaster

• P-elements are transposable DNA that can cause
  hybrid dysgenesis (????).(how? )

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Figure 21-21 hybrid dysgenesis

• The F1 progeny are often sterile, when mating
  females from the M strain with males from the
  P strain.

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• 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.

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

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

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• 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.

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• 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.

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• 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.

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

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CHAPTER 15 The Genetic Code

• Model 6 THE HOUSE MOUSE, Mus musculus

4/22/05
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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.

Mus musculus
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• 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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• Then the embryo enters gastrulat-ion (???), and
  the ICM forms all three germ layers endoderm
  (???) , mesoderm (???), ectoderm (???).
• The first stage in gastrulation is the
  subdivision of the ICM into two cell lays an
  inner hypoblast (???) and an outer epiblast (???).

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• Then a groove called primitive streak (??) 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.

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

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Figure 21-23 overview of mouse embryogenesis
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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.

Mus musculus
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Figure 21-24
127

• 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.

Figure 21-25
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Homologous Recombination Permits the Selective
Ablation of Individual Genes
Mus musculus

• 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.

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• 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.

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• 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).
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• 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.

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• 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.

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• 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.

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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
See the detail in chapter 17
Figure 21-27 imprinting in the mouse