Regulatory Region Size: 50 > 10,000 bp



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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 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



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



Array Analysis: Clustering






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