Regulatory Region Size: 50 > 10,000 bp

Genes

Eukaryotic Protein-Coding Gene Structure

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

• Contained in mRNA

• Spans from start of transcription to start of translation

• Multiple functions – translational efficiency

• Size varies greatly - average > 300 nt (human)

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

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)

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

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

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

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

• 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 evolutionarily-relevant time span to detect
• May be conditional

CNS Midline Cell Development and Transcription Requires Single-minded Function

Ubiquitously-Expressed Sim Transforms Entire CNS into CNS Midline Cells

Gene Regulation

• Regulatory proteins > DNA cis-control elements

• Positive and negative regulation

• Combinatorial regulation > highly specific patterns of spatial, temporal and quantitative expression

Sim:Tgo Binding Sites (CNS Midline Elements - CMEs) are Required for Midline Transcription

• 0.95 kb Toll-lacZ

• -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

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

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

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

Analysis of Midline Transcription by Ectopic Sim Expression: Genetic Approaches

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 twi / twi

• GFP-CyO / GFP-Cyo

Mutant Sorting

• GFP-labeled organisms

• Hand sort with fluorescence

• microscope

• Machine sort

Array Analysis: Clustering