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

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

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)

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 designEvolutionarily assembled in piecesFunctional unit > exons

# exons can vary from 1 > 178 Average # exons/gene – different organisms

Yeast ~1Drosophila 4Human 9

Human genes (mean sizes)Exon size 145 bp

coding non-coding

Introns

Introns vary greatly in sizeMost ~ 50 bp but can be > 15 kbLarge genes – large intronsSmall genes – small introns

Size differs between speciesC. elegans 267 bpDrosophila 487 bpHuman 3,365 bpHuman introns > exons in size

Intron 2Intron 1

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 anythingOther genes may compensate for defect

(redundancy)Double mutant analysis often provides evidence

for this explanationCommon for highly-related genes to be (at least

partially) redundantDefect may be too subtle to detect

Proper assay not usedNeed proper ecological setting and evolutionarily-

relevant time span to detectMay be conditional

CNS Midline Cell Development and Transcription Requires Single-minded Function

Cell division

Wild-type

Cell morphology

Gene expression

sim

Ubiquitously-Expressed Sim Transforms Entire CNS into CNS Midline Cells

Heat shock-sim Rhomboid-lacZ

Uninduced

Induced

-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

0.95 kb Toll-lacZ

-LacZ

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

2 431

X XXX

CME > ACGTG

Array Analysis of Gene Expression: Drosophila

Understand complete array of gene regulatory events that underlie:DevelopmentTissue and cell identityAgingBehavior

Circadian rhythmsLearning and memory

Example: Single-minded (Sim): Master Regulator of CNS Midline Cell Development and Transcription

Sim protein (green) > CNS midline cellsVnd protein (red) > lateral CNS

Array Analysis of Gene Expression

Midline gene expression program > identify all genes expressed in midline cellsStudy: 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-typeTransgenes – express in entire CNSGenetics – snail mutant > express in entire

mesoderm

Midline and Lateral CNS GFP Lines

sim-GFP vnd-GFP

Dissociate embryonic cells > FACSCompare expression at different stages and to other cell typesResults: midline-specific transcripts high in midline cells when comparedto levels in other tissues

Fluorescence Activated Cell Sorter (FACS)

Allows isolation offluorescently-labeled(GFP+) cells

Array Analysis of Gene Expression

Midline gene expression program > identify all genes expressed in midline cellsStudy: 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-typeTransgenes – express in entire CNSGenetics – 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 cellsStudy: 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-typeTransgenes – express in entire CNSGenetics – snail mutant > express in entire

mesoderm

Analysis of Midline Transcription by Ectopic Sim Expression: Transgenic Approaches

sca-Gal4 X UAS-sim-GFP

GFP -Wrapper

Wild-type

-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

snail

sim RNAlocalization

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 musclesVisceral musclesFat body, hemocytes

twist geneEncodes 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

Confirm expected expression pattern by in situ hybridization