Genes

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Genes
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Eukaryotic Protein-Coding Gene Structure
coding
non-coding
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Regulatory Region

• Size 50 gt 10,000 bp
• Contains multiple small DNA sequence elements (5
  20 bp) gt bind regulatory proteins
• Regulatory elements can be negative or positive
  acting
• Regulatory regions found in 5 flanking region,
  introns, and 3 flanking regions most common in
  5 flanking regions and large introns

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5-Untranslated Region

• Contained in mRNA
• Spans from start of transcription to start of
  translation
• Multiple functions translational efficiency
• Size varies greatly - average gt 300 nt (human)

coding
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Coding Sequence

• Begins with initiator methionine (AUG codon)
• Sometimes multiple initiator methionines are used
• Stops with termination codon (UAA, UAG, and UGA)
• Sizes varies average 1340 nt (human) encodes
  450 aa protein

coding
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3 Untranslated Region

• Spans translational termination codon gt end of
  mRNA
• Multiple functions mRNA stability and
  localization
• AAUAAA sequence signals where poly(A) is to be
  added
• (10-35 nt upstream from cleavage/poly(A)
  site)
• Size varies average - 700 nt (human)

coding
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Poly(A)

• Added posttranscriptionally (not encoded in gene)
• Size varies (10-200 nt) depending on organism
• Functions mRNA stability and translational
  efficiency
• Size of tract shortens with time
• All mammalian mRNAs have poly(A) except histone
  mRNAs

Poly(A)
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Exons

• Genes have a modular design
• Evolutionarily assembled in pieces
• Functional unit gt exons
• exons can vary from 1 gt 178
• Average exons/gene different organisms
• Yeast 1
• Drosophila 4
• Human 9
• Human genes (mean sizes)
• Exon size 145 bp

coding
non-coding
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Introns

• Introns vary greatly in size
• Most 50 bp but can be gt 15 kb
• Large genes large introns
• Small genes small introns
• Size differs between species
• C. elegans 267 bp
• Drosophila 487 bp
• Human 3,365 bp
• Human introns gt exons in size

Intron 2
Intron 1
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Genetics

• Mutants
• Wild-type normal fully-active gene
• Null absence of any activity (e.g. deletion)
• Hypomorph reduced function
• Hypermorph enhanced activity
• Neomorph expressed in cells normally not
  expressed (transgenic approach)
• Phenotypic analysis development, morphology,
  behavior, fertility, etc.
• Gene regulation
• Examine how mutation in Gene A influences
  expression of other genes

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Genetic and Molecular Genic Relationships

• Organism Genes
  Lethal loci (total genes)
• Yeast 5,800
  1,800 (30)
• Nematode 18,400
  3,500 (20)
• Drosophila 13,600
  3,600 (25)
• Mouse similar based on gene knockout studies
• Lethal loci loss of function mutant that
  results in death
• Result Only 20-30 genes can be mutated to
  lethality

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Genetic and Molecular Genic Relationships

• Why are there genes with no apparent function?
• Gene may not be doing anything
• Other genes may compensate for defect
  (redundancy)
• Double mutant analysis often provides evidence
  for this explanation
• Common for highly-related genes to be (at least
  partially) redundant
• Defect may be too subtle to detect
• Proper assay not used
• Need proper ecological setting and
  evolutionarily-relevant time span to detect
• May be conditional

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CNS Midline Cell Development and Transcription
Requires Single-minded Function
Wild-type
sim
Cell division
Cell morphology
Gene expression
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Ubiquitously-Expressed Sim Transforms Entire CNS
into CNS Midline Cells
Heat shock-sim
Rhomboid-lacZ
Uninduced
Induced
a-LacZ
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Gene Regulation

• Regulatory proteins gt DNA cis-control elements
• Positive and negative regulation
• Combinatorial regulation gt highly specific
  patterns of spatial, temporal and quantitative
  expression

Murine transthyretin gene
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SimTgo Binding Sites (CNS Midline Elements -
CMEs) are Required for Midline Transcription

• 0.95 kb Toll-lacZ
• 
  a-LacZ

2
4
3
1
X
X
X
X
CME gt ACGTG
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Array Analysis of Gene Expression Drosophila

• Understand complete array of gene regulatory
  events that underlie
• Development
• Tissue and cell identity
• Aging
• Behavior
• Circadian rhythms
• Learning and memory

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Example Single-minded (Sim) Master Regulator of
CNS Midline Cell Development and Transcription
Sim protein (green) gt CNS midline cells Vnd
protein (red) gt lateral CNS
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Array Analysis of Gene Expression

• Midline gene expression program gt identify all
  genes expressed in midline cells
• Study function and regulation
• Approaches
• Purify midline cells (GFP) gt compare to other
  cell types and developmental time intervals
• Mutant (sim) vs. wild-type
• Misexpression of sim vs. wild-type
• Transgenes express in entire CNS
• Genetics snail mutant gt express in entire
  mesoderm

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Midline and Lateral CNS GFP Lines
sim-GFP
vnd-GFP
Dissociate embryonic cells gt FACS Compare
expression at different stages and to other cell
types Results midline-specific transcripts high
in midline cells when compared to levels in other
tissues
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Fluorescence Activated Cell Sorter (FACS)

• Allows isolation of
• fluorescently-labeled
• (GFP) cells

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Array Analysis of Gene Expression

• Midline gene expression program gt identify all
  genes expressed in midline cells
• Study function and regulation
• Approaches
• Purify midline cells (GFP) gt compare to other
  cell types and developmental time intervals
• Mutant (sim) vs. wild-type
• Misexpression of sim vs. wild-type
• Transgenes express in entire CNS
• Genetics snail mutant gt express in entire
  mesoderm

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Comparison of Wild-type to sim Mutant Embryos
Wild-type
sim
Results Expect to see midline gene expression
reduced in sim mutant
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Array Analysis of Gene Expression

• Midline gene expression program gt identify all
  genes expressed in midline cells
• Study function and regulation
• Approaches
• Purify midline cells (GFP) gt compare to other
  cell types and developmental time intervals
• Mutant (sim) vs. wild-type
• Misexpression of sim vs. wild-type
• Transgenes express in entire CNS
• Genetics snail mutant gt express in entire
  mesoderm

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Analysis of Midline Transcription by Ectopic Sim
Expression Transgenic Approaches
sca-Gal4 X UAS-sim-GFP
Wild-type
GFP
a-Wrapper
a-Wrapper
Result Expect to see midline gene expression
increased in sca-Gal4 X UAS-sim-GFP
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Analysis of Midline Transcription by Ectopic Sim
Expression Genetic Approaches
Wild-type
sim RNA localization
snail
Result Expect to see midline gene expression
increased in snail mutant
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Cluster Analysis of Combined Data Sets

• Compare different data sets
• Midline genes
• Test by in situ hybridization for midline
  expression

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Array Analysis of Mesoderm Gene Expression

• Mesoderm
• Somatic muscles
• Visceral muscles
• Fat body, hemocytes
• twist gene
• Encodes transcription factor required for
  mesodermal gene expression
• twist mutant no mesoderm or mesodermal gene
  expression
• twist overexpression (Toll10B mutation) excess
  mesoderm and mesodermal gene expression

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Twist Mutant and Overexpression Phenotypes
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Mutant Embryo Purification

• twist is embryonic lethal mutation
• twi / X twi / only 25 embryos are mutant
  (twi / twi)
• Use GFP-CyO chromosome and sort mutant embryos
• GFP-CyO / twi
  twi / twi
• GFP-CyO / GFP-Cyo
  

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Mutant Sorting

• GFP-labeled organisms
• Hand sort with fluorescence
• microscope
• Machine sort

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Array Analysis Clustering
Confirm expected expression pattern by in situ
hybridization
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