Transcript PAS Meeting - University of North Carolina at Chapel Hill

Genes
Eukaryotic Protein-Coding Gene Structure
coding
non-coding
Regulatory Region
 Size: 50 > 10,000 bp
 Contains multiple small DNA sequence elements (5 – 20 bp) >
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
5’-Untranslated Region
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Contained in mRNA
Spans from start of transcription to start of translation
Multiple functions – translational efficiency
Size varies greatly - average > 300 nt (human)
coding
non-coding
Coding Sequence
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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
non-coding
3’ Untranslated Region
 Spans translational termination codon > 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
non-coding
Poly(A)
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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)
Exons
 Genes have a modular design
 Evolutionarily assembled in pieces
 Functional unit > exons
 # exons can vary from 1 > 178
 Average # exons/gene – different organisms
 Yeast
~1
 Drosophila
4
 Human
9
 Human genes (mean sizes)
 Exon size
145 bp
coding
non-coding
Introns
 Introns vary greatly in size
 Most ~ 50 bp but can be > 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 > exons in size
Intron 1
Intron 2
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
Genetic and Molecular Genic Relationships
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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
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 evolutionarilyrelevant time span to detect
May be conditional
CNS Midline Cell Development and Transcription Requires
Single-minded Function
Wild-type
Cell division
Cell morphology
Gene expression
sim
Ubiquitously-Expressed Sim Transforms Entire CNS into
CNS Midline Cells
Heat shock-sim
Rhomboid-lacZ
Uninduced
Induced
a-LacZ
Gene Regulation
 Regulatory proteins > DNA cis-control elements
 Positive and negative regulation
 Combinatorial regulation > highly specific patterns of spatial,
temporal and quantitative expression
Murine transthyretin gene
Sim:Tgo Binding Sites (CNS Midline Elements - CMEs)
are Required for Midline Transcription
0.95 kb Toll-lacZ
1
2 3
4
X
X X X
CME > ACGTG
a-LacZ
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
Example: Single-minded (Sim): Master Regulator of CNS
Midline Cell Development and Transcription
Sim protein (green) > CNS midline cells
Vnd protein (red) > lateral CNS
Array Analysis of Gene Expression
 Midline gene expression program > identify all genes
expressed in midline cells
 Study: function and regulation
 Approaches:
 Purify midline cells (GFP) > 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 > express in entire mesoderm
Midline and Lateral CNS GFP Lines
sim-GFP
vnd-GFP
Dissociate embryonic cells > 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
Fluorescence Activated Cell Sorter (FACS)
Allows isolation of
fluorescently-labeled
(GFP+) cells
Array Analysis of Gene Expression
 Midline gene expression program > identify all genes
expressed in midline cells
 Study: function and regulation
 Approaches:
 Purify midline cells (GFP) > 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 > express in entire mesoderm
Comparison of Wild-type to sim Mutant Embryos
Wild-type
sim
Results: Expect to see midline gene expression reduced in sim mutant
Array Analysis of Gene Expression
 Midline gene expression program > identify all genes
expressed in midline cells
 Study: function and regulation
 Approaches:
 Purify midline cells (GFP) > 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 > express in entire mesoderm
Analysis of Midline Transcription by Ectopic Sim
Expression: Transgenic Approaches
Wild-type
sca-Gal4 X UAS-sim-GFP
a-Wrapper
GFP
a-Wrapper
Result: Expect to see midline gene expression increased in sca-Gal4 X UAS-sim-GFP
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
Cluster Analysis of Combined Data Sets
 Compare different data sets
 Midline genes
 Test by in situ hybridization for midline expression
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
Twist Mutant and Overexpression Phenotypes
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
GFP-CyO / GFP-Cyo
twi / twi
Mutant Sorting
 GFP-labeled organisms
 Hand sort with fluorescence
microscope
 Machine sort
Array Analysis: Clustering
Confirm expected expression pattern by in situ hybridization