Gene Expression

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Gene Expression
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Gene expression

• All cells in one organism have the same DNA. But
  different cells have very different functions.
• In each cell at certain times only some genes are
  expressed.
• Which genes are expressed at which times?

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Cells

• muscle

• nerve

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Double-stranded DNA
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DNA Structure
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DNA matching

• Every A forms two weak hydrogen bonds with T.
• Every T forms two hydrogen bonds with A.
• Every C forms three weak hydrogen bonds with G.
• Every G forms three hydrogen bonds with C.

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RNA

• RNA is also a sequence of nucleotides.
• RNA means ribonucleic acid.
• DNA means deoxyribonucleic acid.

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Nucleotides
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RNA
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DNA Structure
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DNA vs RNA

• Both are strings of nucleotides.
• DNA is usually double-stranded RNA is
  single-stranded.
• RNA is usually much shorter than DNA.
• RNA replaces each T by U (uracil).
• DNA contains deoxyribose while RNA contains
  ribose. This makes DNA more stable chemically
  than RNA.

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DNA and RNA

• DNA in your cells is in the nucleus RNA can be
  anywhere in the cell.
• Proteins are made directly using RNA, not DNA.

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Central Dogma

• A protein-coding region of DNA is copied to
  messenger RNA (mRNA) by transcription.
• The mRNA leaves the nucleus and goes to a
  ribosome.
• The ribosome uses the mRNA to make a protein by
  translation.

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Central Dogma
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Translating codons

• Ala/A GCT, GCC, GCA, GCG Leu/L TTA, TTG, CTT,
  CTC, CTA, CTG
• Arg/R CGT, CGC, CGA, CGG, AGA, AGG Lys/K AAA, AAG
• Asn/N AAT, AAC Met/M ATG
• Asp/D GAT, GAC Phe/F TTT, TTC
• Cys/C TGT, TGC Pro/P CCT, CCC, CCA, CCG
• Gln/Q CAA, CAG Ser/S TCT, TCC, TCA, TCG, AGT,
  AGC
• Glu/E GAA, GAG Thr/T ACT, ACC, ACA, ACG
• Gly/G GGT, GGC, GGA, GGG Trp/W TGG
• His/H CAT, CAC Tyr/Y TAT, TAC
• Ile/I ATT, ATC, ATA Val/V GTT, GTC, GTA, GTG
• START ATG STOP TAG, TGA, TAA

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Protein primary structure
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3D views of proteins
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DNA for beta hemoglobin

• ATGGTGCATCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGG
  CAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGCTGCTGG
  TGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGATCTGTCC
  ACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGGCAAGAA
  AGTGCTCGGTGCCTTTAGTGATGGCCTGGCTCACCTGGACAACCTCAAGG
  GCACCTTTGCCACACTGAGTGAGCTGCACTGTGACAAGCTGCACGTGGAT
  CCTGAGAACTTCAGGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCA
  TCACTTTGGCAAAGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAG
  TGGTGGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAA

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Primary structure for beta hemoglobin

• MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYWTQRFFESFGDLST
  PDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDP
  ENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH

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Part of the two strands for beta hemoglobin

• ATGGTGCATCTGACTCCT
• TACCACGTAGACTGAGGA
• The top is the sense or template the bottom is
  the antisense or coding strand.

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Transcription Make mRNA

• ATGGTGCATCTGACTCCT sense
• TACCACGTAGACTGAGGA coding
• AUGGUGCAUCUGACUCCU mRNA

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Structure of mRNA
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mRNA goes to a ribosome, outside the nucleus

• AUGGUGCAUCUGACUCCU mRNA

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Eukaryotic cell

• (1) nucleolus
• (2) nucleus
• (3) ribosomes (little dots)
• (5) rough endoplasmic reticulum (ER)
• (9) mitochondria
• (10) vacuole
• (11) cytoplasm

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Ribosomes

• The ribosome functions as a factory to make
  proteins. It uses two kinds of input
• (a) mRNA
• (b) tRNA
• It outputs a protein.

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Ribosome translates mRNA

• Ribosome (2) straddles mRNA (1)
• It makes the protein (3).
• It starts at AUG and ends at UAG

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Ribosome large subunit
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Transfer RNA (tRNA)

• Each tRNA molecule has on one side a conformation
  that binds to the specific codon and on the other
  side a conformation that binds to the
  corresponding amino acid.

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tRNA

• CCA tail in orange, Acceptor stem in purple, D
  arm in red, Anticodon arm in blue with Anticodon
  in black, T arm in green.

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tRNA carries the amino acid matched to the codon

• UAC M tRNA will bind with the codon AUG
  in the mRNA.
• CAC V tRNA will bind with the codon GUG in
  the mRNA.

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mRNA in a ribosome has the genetic information

• AUGGUGCAUCUGACUCCU
• UAC M tRNA will bind with the codon AUG.
• CAC V tRNA will bind with the codon GUG.

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Translating codons

• Ala/A GCT, GCC, GCA, GCG Leu/L TTA, TTG, CTT,
  CTC, CTA, CTG
• Arg/R CGT, CGC, CGA, CGG, AGA, AGG Lys/K AAA, AAG
• Asn/N AAT, AAC Met/M ATG
• Asp/D GAT, GAC Phe/F TTT, TTC
• Cys/C TGT, TGC Pro/P CCT, CCC, CCA, CCG
• Gln/Q CAA, CAG Ser/S TCT, TCC, TCA, TCG, AGT,
  AGC
• Glu/E GAA, GAG Thr/T ACT, ACC, ACA, ACG
• Gly/G GGT, GGC, GGA, GGG Trp/W TGG
• His/H CAT, CAC Tyr/Y TAT, TAC
• Ile/I ATT, ATC, ATA Val/V GTT, GTC, GTA, GTG
• START ATG STOP TAG, TGA, TAA

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mRNA goes to a ribosome

• AUGGUGCAUCUGACUCCU mRNA
• UAC . M tRNA
• CAC V tRNA
• The ribosome matches UAC on tRNA with AUG on
  mRNA, then uses the M on the other end in the
  protein.

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mRNA goes to a ribosome

• AUGGUGCAUCUGACUCCU mRNA
• UAC . M tRNA
• CAC V tRNA
• The ribosome matches CAC on tRNA with GUG on
  mRNA, then uses the V on the other end to extend
  the protein.

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Ribosome

• In this manner, the ribosome continues to make
  the protein until it reaches a STOP codon.

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When is a given gene being expressed?

• A given protein is being made when its mRNA is
  present in the cell.
• The DNA is always present.

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When is a given gene being expressed?

• To tell what is being expressed at a given time
  in a given cell, find out which mRNAs are
  present.
• For each kind of mRNA, measure the quantity
  present.

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A microarray
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Microarrays

• A microarray consists of a pattern of thousands
  of features.
• Each feature has some DNA that will probe and
  possibly bind with an mRNA sample.
• Typically the feature is made to fluoresce under
  the presence of binding mRNA.
• The brightness of the dot corresponds to the
  quantity of mRNA of the given sort that is
  present.

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Two gene chips
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Microarrays

• Typically the probe is attached to a solid
  surface which is a glass or silicon chip. It is
  then called a gene chip or Affymetrix microarray.

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Introns

• Introns are inserts in the DNA within portions
  that code for one protein.
• The parts that code are exons.

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Introns must be removed to make the mature mRNA
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cDNA

• Complementary DNA (cDNA) is DNA synthesized from
  mature mRNA using reverse transcriptase.
• AUGGUGCAUCUG mRNA
• TACCACGTAGAC cDNA

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cDNA

• cDNA is more stable than RNA.
• cDNA corresponds with the part of the genome from
  which introns have been removed.
• cDNA does not correspond exactly to nuclear DNA.

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The mature mRNA
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The probes

• Each dot can contain DNA, cDNA, or an
  oligonucleotide (oligo).
• An oligonucleotide is a short fragment of
  single-stranded DNA, typically 5 to 50
  nucleotides long.

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Gene expression profiling

• In an mRNA or gene expression profiling
  experiment the expression levels of thousands of
  genes are monitored simultaneously in parallel.
  This can be used to distinguish
• (a) the effects of certain treatments
• (b) the effects of diseases
• (c) the effects of different stages of
  development.

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Gene expression profiling

• For example, microarrays can identify genes whose
  expression is changed in response to pathogens
  by comparing gene expression in infected cells to
  that in uninfected cells.

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A microarray experiment

• Suppose there are two cells--type 1, healthy, and
  type 2, diseased. Both have four genes A, B, C,
  D. We want to compare the expression of these
  genes in the two types of cell.

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Procedure

• 1. Prepare the DNA chip using the chosen target
  DNAs.
• 2. From the cells, isolate the mRNA.
• 3. Use the mRNA as templates to generate cDNA
  with a fluorescent tag attached. Typically a
  green fluorescent tag is used for mRNA from
  healthy cells, while a red tag is used for mRNA
  from diseased cells.

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Procedure

• 4. Prepare a hybridization solution with a
  mixture of the fluorescently labeled cDNAs.
• 5. Incubate the hybridization solution with the
  DNA chip.
• 6. Detect bound cDNA using laser technology.
• 7. Analyze the data.

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Appearance afterwards
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Interpreting colors

• A spot with just healthy cDNA is green.
• A spot with just diseased cDNA is red.
• A spot with both is yellow.
• A spot with neither is black.

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Comparison of cells

• Microarrays are used to compare the genome
  content in different cells for the same organism.

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Single Nucleotide Polymorphisms

• A single nucleotide polymorphism is a single
  substitution in the genome.
• Example
• AUGGUGCAUCUGACUCCU standard
• AUGGUGUAUCUGACUCCU SNP

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Detecting SNPs

• Microarrays can be used to detect SNPs between or
  within populations.
• This can measure predisposition to diseases or
  identify appropriate drugs.

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How are chips made?

• In spotted microarrays the probes may be small
  fragments of DNA. An array of fine needles is
  controlled by a robotic arm that is dipped into
  wells containing the DNA probes. Each needle
  then deposits a probe at the desired location on
  the surface. The probes are fixed to the
  surface. Then the chip is ready to be washed in
  a solution containing the targets.

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DNA microarray being printed by a robot
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Flexibility of microarrays

• Thus scientists can produce arrays from their own
  labs, customized to an experiment.

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Bioinformatics problems

• 1. How long should the probes be (i.e., how many
  nucleotides long)?
• If too short, you get false signals.
• If too long, it is expensive.

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Bioinformatics problems

• 2. Which parts of a sequence should be cloned in
  the probe?

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DNA for beta hemoglobin

• ATGGTGCATCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGG
  CAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGCTGCTGG
  TGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGATCTGTCC
  ACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGGCAAGAA
  AGTGCTCGGTGCCTTTAGTGATGGCCTGGCTCACCTGGACAACCTCAAGG
  GCACCTTTGCCACACTGAGTGAGCTGCACTGTGACAAGCTGCACGTGGAT
  CCTGAGAACTTCAGGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCA
  TCACTTTGGCAAAGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAG
  TGGTGGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAA

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Statistical issues in microarrays

• 1. There is variability in how well each probe
  in the microarray was made.
• 2. There is variability in how uniformly the
  target got washed across the chip.
• 3. There is variability in how accurately the
  probe binds with the target.

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Statistical questions

• What level of expression is statistically
  significant?
• If there are 20,000 probes, a 95 confidence
  means there are ? events with probability less
  than 5.

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Statistical questions

• What level of expression is statistically
  significant?
• If there are 20,000 probes, a 95 confidence
  means there are 1000 events with probability less
  than 5.

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Statistical issues

• How can the data be normalized (ie, compared with
  known probability distributions, like the normal
  curve)?
• P values there will be false positives and
  false negatives.

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Experimental design issues

• Replication of biological samples
• Replication of RNA samples from each experiment
• Replicate each spot on the microarray

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Data warehousing

• The data bases are huge hence hard to understand.