PowerPoint to accompany
Genetics: From Genes to Genomes
Fourth Edition
Hartwell ● Hood ● Goldberg ● Reynolds ● Silver
Reference
D
Prepared by Malcolm Schug
University of North Carolina Greensboro
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D-1
Drosophila melanogaster:
Genetic Portrait of the Fruit Fly
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Fig. D.1
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Outline of Reference D
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Structure and organization of genome
Life cycle
Techniques of genetic analysis
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Balancer chromosomes
P-element transposons
Production of mosaics
Ectopic gene expression
Targeted gene knockouts
Drosophila genome project
Genetic analysis of body plan development in
Drosophila: a comprehensive example
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Structure of the Drosophila Genome
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Chromosomes of
Drosophila
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Fig. D.3
Four chromosomes
designated 1-4
XY sex
determination (XX
females, XY males)
Sex determined by
X:A ratio
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Giant polytene
chromosomes of larval
salivary gland are key
tools
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Fig. 14.6a
Replicate 10-11 times
1024-2048 sister chromatids
stay associated under
perfect lateral register
Homologous chromosome
stay tightly synapsed
Chromocenter – common
region where centromeres
coalesce
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Drosophila Genome
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170,000 kb of DNA
21% is highly repetitive satellite DNA in
heterochromatin and Y chromosome
3% repeated genes for rRNA, 5s RNA and
histones
9% is 50 families of transposons 2-9 kb in length
Telomeres do not have simple repeats
Telomeres have transposable element sequences
67% of genome-unique sequences in euchromatin
comprising about 13,600 genes
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Life cycle
Fig. D.4
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Many Adult Structures Develop from
Imaginal Discs in Larvae and Pupae
Fig. D.5
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Balancer Chromosomes help
Preserve Linkage
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Fig. D.6
Balancers carry
multiple, overlapping
inversions
Most contain a
dominant marker and
recessive lethal
mutation that
prevents survival of
homozygotes
Useful in many
genetic experiments
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Techniques of Genetic Analysis
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In Drosophila, crossing over occurs only in
females
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Absence of crossing over in males has considerable
technical significance
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Maintain linkage relationships by inheritance through male
parent
In females, only a moderate amount of crossing over
occurs
1-2 crossovers per meiosis
 No crossing over in heterochromatin or 4th chromosome
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P-element Transposons are Critical
Tools in Molecular Genetics
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Hybrid dysgenesis
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Males from Drosophila strains carrying P elements crossed to
females that lack P elements
P element becomes highly mobile in germ line of F1 hybrids
Chromosome breakage reduces fertility in hybrids
Progeny of F1 flies carry many new mutations induced by P
element insertions
Molecular details
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P element primary transcript encodes transposase that catalyzes
transposition
Cross between P and M strain causes hybrid dysgenesis
Cross between P and P strain does not
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Eggs produced by P female have repressor protein that prevents
transposition
Repressor coded for by alternatively spliced P element mRNA
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Fig. D.7
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Transformation: the Introduction of
Cloned DNA into Flies
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Fig. D.8a
P elements used as
vectors
Insert fly DNA into
intact P element and
then into plasmid
Inject into syncytial
embryos from M strain
mothers
Cross to P males
Mimicking hybrid
dysgenesis
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P Element Tagging Helps Clone
Genes of Developmental Interest
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P element contains visible marker such
as white and plasmid with antibiotic
resistance
Plasmid rescue – cut DNA from
mutant strain and circularize with
DNA ligase
Transform into E. coli
Plasmid plus gene-containing fly DNA
flanking P element insertions
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Isolation of a DNA Sequence from a
Transposon-tagged Gene
Plasmid Rescue
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Isolation of a DNA Sequence from a
Transposon-tagged Gene
Inverse PCR
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Enhancer Trapping to Identify
Genes by Expression Pattern
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Fig. D.10
P element with lacZ gene
downstream of promoter
When mobilized, 65% of new
insertions express lacZ
reporter during development
Promoter can only activate
transcription if under control
of enhancers of genes near
insertion site
Detects genes turned on in
certain tissues
Genes isolated by plasmid
rescue
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Genetic Mosaics
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Fig. D.12 a
Composed of cells of more
than one genotype
Generated by mitotic
recombination
X-ray induced mutations
cause crossing-over in
homologues in somatic cells
X-ray method has now
been replaced by sitespecific recombination
enzyme from yeast called
FLP
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Using Mosaics to Determine if Genes Needed for
Embryo Viability Also Play a Role Later in
Development
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What if a mutant gene arrests development early when homozygous?
How do you study its effects later in development?
Mosaics produced by FLP, a site-specific recombination enzyme from
yeast
Create genes homozygous in adult that are heterozygous in early
development
Fig. D.11
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Testing Ability of Cell and its Descendents to
Contribute to Adult Structures
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Minute mutations
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Fig. D.12 b
50 minute genes throughout
genome
Heterozygotes cause reduced
rate of protein synthesis
Mitotic recombination
produces Minute+/Minute+
cells in Minute+/Minutebackground
+/+ cells will grow big
Determines fate of cells by
examining tissue growth
after mitotic recombination
event
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Mosaics Used to Tell When and Where a Gene Must
be Expressed to Generate Normal Phenotype
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Fig. D.13 a,b
Ommatidia are facets
of fly’s compound eye
During development
undifferentiated cells
are recruited to
become photoreceptors (R1-R8) for
each facet
Final photoreceptor in
each group is R7
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Sevenless (sev) and bride of sevenless (boss) mutants
develop without R7
Mosaics generated with marked cells (white)
Requirement for Sev+ is cell autonomous
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Fig. D.13c
Presumptive R7 cell must make Sev+ protein
Sev+ affects only cell in which it is made
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R7 cell develops properly only if R8 cell makes Boss+
Boss+ is not cell autonomous
 Produced in one cell (R8) but affects differentiation of
different cell (R7)
Fig. D.13d
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Using Mosaics to Determine Importance
of Gene Product for Oogenesis
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Expression outside of cells or tissue where gene is normally expressed
Eyeless protein under control of heat shock promoter and introduce
into genome by P element transposition
Eyes appear anywhere on fly’s body when heat shocked
Fig. D.15
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The Drosophila Genome Project
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13,600 known or predicted genes present
One gene every 9 kb
Half of fly proteins homologous with mammalian
proteins
One third homologous to nematodes
61% of human disease genes have homologues in
flies
30% of genes unrelated to genes in other
organisms
Only 4000 genes essential for viability
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A New Direction for Drosophila Genome
Project: Obtaining Mutations in All Fly Genes
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Isolate as many P element insertion alleles
as possible of as many genes as possible
To date – P element disruptions for 65% of
genes
New methods of gene knockout such as
RNAi to circumvent difficulties with
knockouts by homologous recombination
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Genetic Analysis of Body Plan Development:
a Comprehensive Example
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How body becomes specialized along both anteriorposterior (AP) and dorsal-ventral (DV) axes
Segmentation genes subdivide body into an array of
identical body segments
Homeotic genes assign a unique identity to each body
segment
How does the developing animal establish the proper
number of body segments?
How does each body segment know what kinds of
structures it should form and what role it should play in
the animal’s body?
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Fig. D.18
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Fig. D.19
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Replace d. with fig
D.19d from 3e
Four Classes of Genes Responsible
for Formation of Segments
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Maternal genes
Gap genes
Pair-rule genes
Segmentation polarity genes
Function in a hierarchy that progressively
subdivides the embryo into successively
smaller units
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Maternal Genes Interact to Produce
Morphogen Gradients
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Maternal-effect mutations
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Recessive mutations in maternal genes that influence embryonic
development
Maternally supplied components account for formation of
body plan between fertilization and end of 13 syncytial
divisions
Nusslein-Volhard and Wieschaus screened thousands of
mutagen treated chromosomes by examining phenotypes of
embryos from homozygous mutant mothers
Focuses on stocks with homozygous mutant sterile females
Identified large number of maternal genes
Nobel Prize in medicine
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Morphogen – substances that define
different cell fates in a concentrationdependent manner
Interaction of two signaling centers located
in the anterior and posterior poles of the egg
pattern insect body axis
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bicoid (bcd) Encodes the Anterior
Morphogen
Image seems blurry – replace with
Figure D.20 a and c if possible –
if not delete this text box.
Fig. D.20 a,c
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How Bcd Protein Works
Figure D. 21
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Fig. D.21
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Specification of Segment Number through
Activation of Zygotic Genes in Successively More
Sharply Defined Regions of the Embryo
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Zygotic genes
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Nusslein-Volhard and Wieschaus performed zygotic
mutant screen
Ethyl methane sulfonate (EMS) mutagenesis
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Homozygous mutant embryos
Recessive lethals
Three classes of genes
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Transcribed and translated from DNA in nuclei of embryonic cells
Expression begins in syncytial blastoderm stage (roughly cycle 10)
9 gap genes
8 pair-rule genes
17 segment-polarity genes
Hierarchy of gene expression
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Gap genes
Expressed first
 Gap mutants show a gap in segmentation
pattern at positions where particular gene is
absent
 Binding sites in promoter have different
affinities for maternal transcription factors
 Gap genes encode transcription factors that
influence expression of other gap genes
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Zones of Expression of Four Gap Genes:
Hunchback, Kruppel, Knirps, and Giant in
Late Syncytial Blastoderm Embryos
Figure D.22a
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Defects in Segmentation from Mutations in
Gap Genes
B)
C)
D)
A)
Fig. D.22b
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Mutations in Gap Gene Result in Loss of Segments
Corresponding to Zone of Expression
Fig. D.22 c
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Segment Polarity Genes are Lowest
Level of Segmentation Hierarchy
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Mutations in segment polarity genes cause
deletion of part of each segment and its
replacement by mirror image of different part of
next segment
Regulatory system complex
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Transcription factors encoded by pair-rule genes
initiate pattern by regulating segment polarity genes
Interactions between cell polarity genes maintain
periodicity later in development
Activation occurs after cellularization of embryo is
complete
Diffusion of transcription factors within syncytium
ceases to play a role
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Pair-rule Genes
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(a) zones of expression at
beginning of blastoderm
stage
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Fig. D.23
Each gene expressed in seven
stripes
(b) formation of Eve stripe
2 requires activation by
Bcd and Hb proteins and
repression by Gt and Kr
proteins
700 bp upstream
regulatory region of eve
gene that directs the Eve
second stripe contains
multiple binding sites
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Fig. D.24
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Fig. D.25
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Each Segment Establishes own Identity Through
Activation of Homeotic Genes
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Homeotic mutations
cause different
segments to develop as
if located elsewhere
bithorax (bx)
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Fig. D.26
Anterior third thoracic
segment (T3) develops
like second anterior
thoracic segment (T2)
postbithorax (pbx)
posterior T3 transforms
into posterior T2
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Figure D.28
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Antennapedia Complex and the
Homeobox
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Homeotic selector
genes
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Fig. D.27
Two clusters of genes on
third chromosome –
antennapedia complex
and bithorax complex
Responsible for
determining segment
identity
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Antennapedia Complex and the Homeobox
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Specifies the identities of segments in head and
anterior thorax
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Five genes
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labial (lab) – expressed in intercalary region
proboscipedia (pb) expressed in maxillary and labial segments
Deformed (Dfd) – expressed in mandibular and maxillary
segments
Sex combs reduced (Scr) – expressed in labial and T1 segments
Antennapedia (Antp) – expressed mainly in T2, but also active
in lower levels in all three thoracic segments and abdomen
Other genes (not homeotic)
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zerknullt (zen) – specifies dorsal embryonic structures
fushi tarazu (ftz) – segmentation gene of pair-rule class
bicoid (bcd) – encodes maternally supplied anterior
determinant
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