Prokaryote and Eukaryote

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Prokaryote and Eukaryote
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Regulation of Gene Expression

• Transcriptional Regulation
• Post-transcriptional Regulation

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Transcriptional Regulation

• Cis factors
• DNA sequences in the vicinity of the structural
  portion of a gene that are required for gene
  expression
• Trans factors
• - factors, usually considered to be proteins,
  that bind to the cis-acting sequences to control
  gene expression

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Transcriptional Regulation

• Cis factors
• Promoters

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-10
-35
-10, TATAAT -35, TTGACA
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Transcriptional Regulation

• Cis factors
• Operators

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-10
-35
-35
-10
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Transcriptional Regulation

• Cis factors
• Activator or enhancer binding sites

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-10
-35
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-10
-35
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Transcriptional Regulation

• Trans factors
• Activators
• Repressors
• Inducers chemicals, proton,
• Apo-repressors
• Co-repressors

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Assay for DNA-Protein Interactions

• Filter Binding

Nitrocellulose membrane
Binds single-stranded DNA, but not double
stranded DNA Binds Protein-DNA complex
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Assay for DNA-Protein Interactions

• Gel mobility shift

Antibody
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Assay for DNA-Protein Interactions

• Footprinting
• DNase
• OH.-

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Tools to Analyze Gene Expression

• In vivo approaches
• Gene fusion and operon fusion
• Reporter genes
• In vitro approaches
• Northern blotting
• Primer extension
• In vitro transcription

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Reporter Genes

• Enzymes that are easy to assay
• b-galactosidase (E. coli)
• Chloramphenicol acetyltransferase (E. coli)
• Alkaline phosphatase
• Proteins that can emit light
• Green fluorescent protein (Jelly fish)

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b-galactosidase Assay
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Chloramphenicol acetyltransferase
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In vitro Tools for Studying Gene Expression

• Northern blotting
• Primer extension
• In vitro transcription

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One at a time is not enough(Micro-Array
Technology)

• Micro-scale
• Global approach
• From bacteria to humans

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How does DNA Micro-Array work?

• Make targets
• Immobilized nucleic acid on micro-array surface
• Make probe
• labeled sample
• Assay (hybridization.)
• Readout
• Informatics (computation)

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Two Types of Micro-Arrays

• Synthetic oligo-nucleotides are spotted on a
  glass slide
• For E. coli genome, each slide (1x1 cm) will have
  over 4000 spots.
• Oligo-nucleotides are directly synthesized on the
  slides
• Very complicate process
• Alpha-matrix company made the first slide.
• Very expensive.

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Prepare Probes

• Prepare high quality mRNA from organisms
• Prepare fluoresce-labeled cDNA using reverse
  transcriptase
• Label the cDNA using fluoresce probes
• Chromophores molecules that are able to absorb
  light.
• Fluorophores molecules that are able to absorb
  and emit light.

mRNA
Reverse transcriptase (RT)
(DNA)
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Fluoresce-label

• Prepare fluoresce-labeled cDNA

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Design a DNA Micro-Array
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Readout and Interpretation
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Results from DNA Micro-Array
What genes are activated? What genes are
repressed? How many folds of the gene expression
are increased or decreased? What is the
relationship between the activated (repressed)
genes?
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Applications of Micro-Array

• Gene discovery
• Disease diagnosis
• Drug discovery Pharmacogenomics
• Toxicological research Toxicogenomics

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Post-Transcriptional Regulation

• Initiation of translation
• Antisense RNA
• Stability of mRNA
• RNA binding proteins
• Rnases activities

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Post-Translational Regulation

• Proteins are modified
• Phosphorylation, nitrotrosylation
• Co-factor assembly
• Half-life time of proteins
• Proteolysis