Genes and Expression

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Genes and Expression

• 51123
• Terry Braun

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Today's Outline

• Gene structure
• genomic structure vs mRNA structure
• ESTs
• coding and noncoding exons
• introns
• primary transcript processing
• memory pneumonic
• alternative splicing and differential
  polyadenylation

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Genome (3 Bb) zoom in
Adenine Thymine Guanine Cytosine ATGC purines
AG pyrimidines CT
www.ensembl.org
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Central Dogma

• gene portions of a genome that affect the
  transcription, translation, and expression of
  functionally active molecules (proteins, DNA
  promoters rRNA, mRNA, tRNA, etc)
• gene often used to describe the coding
  regions of genomes the portions of DNA that are
  made into a protein (via transcription, and
  translation)
• DNA - pre-mRNA - mRNA - protein

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Central Dogma

• DNA - pre-mRNA - mRNA - protein
• DNA is transcribed into pre-mRNA
• introns are removed
• lariat structure
• exons remain (spliced together), also called
  the coding regions called mRNA
• splice site junctions
• mRNA is translated into protein

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Schellenberg MJ, Ritchie DB, MacMillan
AM. Pre-mRNA splicing a complex picture in
higher definition. Trends Biochem Sci. 2008
Jun33(6)243-6. Epub 2008 May 9. Review.
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Gene Structure gene to protein
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Example of Gene in Genomic Context
Context of gene BBS4 in the human
genome. Scale 72.28 Kb Exons and introns Note
possible upstream gene, on other strand Less
than 3 of the genome is transcribed and
translated into a protein.
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Human Genome Project

• Problem
• How do you find all of the genes in a sea of DNA?

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Wheres the gene?

• BBS4 exon2
• TAAAGTAACTCTATCACAATATGGATTTAATGGATTAATTGCATAATTGG
  TGAGCTACTG
• ATTATTCTTGTTATTTGGATGCTTCTTTAAGTTAGCAAGTTTATATTGTG
  GTGCTTCAAT
• ATAGACTACTTATTTCATTTCAGAGAACTCAATTTCCTGTATCTACTGAG
  TCTCAAAAAC
• CCCGGCAGAAAAAAGGTCTGTATGCAGTTTCATGGTATGTGTATGTTTGC
  ACAGACAGAT
• TTCTCTTTTATTTATTTATTTATTTTTTTTTTTGGAGGCAGAGTCTCACT
  GTCACCCAGG
• CTGGAGTGCAGTAGCACAATCTTGGCTCACTGCAACCTTTGCCTCTGGGG
  CTCAAGCAAT
• TCTCCTGCCTCAGCCTCCCGAGTAGCTGGGATTACAGGTGCACGCCACCA
  CACCTGGCTA

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Wheres the gene?

• BBS4 exon2
• TAAAGTAACTCTATCACAATATGGATTTAATGGATTAATTGCATAATTGG
  TGAGCTACTG
• ATTATTCTTGTTATTTGGATGCTTCTTTAAGTTAGCAAGTTTATATTGTG
  GTGCTTCAAT
• ATAGACTACTTATTTCATTTCAGAGAACTCAATTTCCTGTATCTACTGAG
  TCTCAAAAAC
• CCCGGCAGAAAAAAGGTCTGTATGCAGTTTCATGGTATGTGTATGTTTGC
  ACAGACAGAT
• TTCTCTTTTATTTATTTATTTATTTTTTTTTTTGGAGGCAGAGTCTCACT
  GTCACCCAGG
• CTGGAGTGCAGTAGCACAATCTTGGCTCACTGCAACCTTTGCCTCTGGGG
  CTCAAGCAAT
• TCTCCTGCCTCAGCCTCCCGAGTAGCTGGGATTACAGGTGCACGCCACCA
  CACCTGGCTA

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ESTs

• Expressed Sequence Tags
• If we could read the sequence at only the front
  (5') or end(3') of mRNAs (transcripts), or even
  in the middle, that would be conclusive evidence
  of a gene
• Uniquely (?) identify all of the genes
• Do not have full expense of sequencing the whole
  gene sequence (100's of nucleotides VS 1000's)
• Can observe differences of expression in tissues
• Many questioned whether the complete genome
  should even be sequenced

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ESTs at Iowa

• Approach
• Harvest mRNAs and sequence them
• Subtract out what you have already seen (serial
  subtraction)
• Rat gene discovery at Iowa (2003)
• 233,890 3-prime ESTs, 50,075 5-prime ESTs
• 57,822 clusters (8/26/2003)
• novelty 57,822/(233,890 50,075) 0.20

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(No Transcript)
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C-Value ParadoxHartl, Molecular melodies in
high and low C, Nat. Rev. Genetics, Nov 2001

• refers to the massive, counterintuitive and
  seemingly arbitrary differences in genome size
  observed in eukaryotic organisms
• Drosophila melanogaster 180 Mb
• Podisma pedestris 18,000 Mb
• difference is difficult to explain in view of
  apparently similar levels of evolutionary,
  developmental, and behavioral complexity
• more to a genome than coding sequences
• example Alu repeats 250 nucleotides
• humans, chimps, gorillas
• Not in rat/mouse

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Repetitive Elements
number elements

• LINEs 20.4 868,000
• SINEs 13.4 1,558,000
• alus 10.6 1,090,000
• transposons 2.8 294,000
• Sudbery 2002 Human Mol Genetics

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Alternative Splicing

• Every conceivable pattern of alternative
  splicing is found in nature. Exons have multiple
  5 or 3 splice sites alternatively used (a, b).
  Single cassette exons can reside between 2
  constitutive exons such that alternative exon is
  either included or skipped ( c ). Multiple
  cassette exons can reside between 2 constitutive
  exons such that the splicing machinery must
  choose between them (d). Finally, introns can be
  retained in the mRNA and become translated.
• Graveley, Alternative splicing increasing
  diversity in the proteomic world. Trends in
  Genetics, Feb., 2001.

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Each amino acid contains an "amine" group (NH3)
and a "carboxy" group (COOH) (shown in black in
the diagram).The amino acids vary in their side
chains (indicated in blue in the diagram).The
eight amino acids in the orange area are
nonpolar/ hydrophobic.The other amino acids are
polar/ hydrophilic ("water loving").The two
amino acids in the purple box are acidic
("carboxy" group in the side chain).The three
amino acids in the blue box are basic ("amine"
group in the side chain). Know relationship
between DNA, mRNA, and aas
Relevance to disease changes L to I? Cysteine
and disulfide bonds nonpolar internal, polar
external (interacts with H20)
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• A ala alanine M met methionine
• C cys cysteine N asn aspargine
• D asp aspartic acid P pro proline
• E glu glutamic acid Q gln glutamine
• F phe phenylalanine R arg arginine
• G gly glycine S ser serine
• H his histidine T thr threonine
• I ile isoleucine V val valine
• K lys lysine W trp tryptophane
• L leu leucine Y tyr tyrosine

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Review

• Codon Table

degenerate code
gene prediction
One codon Met, Trp. Two codons Asn, Asp, Cys,
Gln, Glu, His, Lys, Phe, Tyr, Three codons Ile,
STOP ("nonsense"). Four codons Ala, Gly, Pro,
Thr, Val. Five codons none. Six codons Arg,
Leu, Ser.
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Mutations

• Mis-sense
• Non-sense
• www.hgvs.org
• http//www.hgvs.org/mutnomen/

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From Slide 6
ATG CCC TTC TCC AAC AGC GT -- splice
donor M P F S N S CCT
GCC CCC CAT GCC TGA P A P H
A STOP Delete CC ATG CCC TTC TAA CAG CCC
M P F Stop Q P TGC CCC CCA TGC
CTG AGG GGC C P P C L
R G ?
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Codon Bias

• PAM1 (Point Accepted Mutations) Dayhoff 1978
• global alignment of closely related proteins (85
  identical)
• Blosum62 (Blocks Substitution Matrix) Henikoff
  1992
• proteins across species containing blocks of
  homology with at least 62 percent were compared
• a residue change measurement was computed based
  on observed residue changes
• rare change -4
• common change 11

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Matrix made by matblas from blosum62.iij
column uses minimum score BLOSUM Clustered
Scoring Matrix in 1/2 Bit Units Blocks
Database /data/blocks_5.0/blocks.dat Cluster
Percentage 62 Entropy 0.6979, Expected
-0.5209 A R N D C Q E G H I L K
M F P S T W Y V B Z X A 4 -1 -2 -2
0 -1 -1 0 -2 -1 -1 -1 -1 -2 -1 1 0 -3 -2 0 -2
-1 0 -4 R -1 5 0 -2 -3 1 0 -2 0 -3 -2 2
-1 -3 -2 -1 -1 -3 -2 -3 -1 0 -1 -4 N -2 0 6
1 -3 0 0 0 1 -3 -3 0 -2 -3 -2 1 0 -4 -2 -3
3 0 -1 -4 D -2 -2 1 6 -3 0 2 -1 -1 -3 -4
-1 -3 -3 -1 0 -1 -4 -3 -3 4 1 -1 -4 C 0 -3
-3 -3 9 -3 -4 -3 -3 -1 -1 -3 -1 -2 -3 -1 -1 -2
-2 -1 -3 -3 -2 -4 Q -1 1 0 0 -3 5 2 -2 0
-3 -2 1 0 -3 -1 0 -1 -2 -1 -2 0 3 -1 -4 E
-1 0 0 2 -4 2 5 -2 0 -3 -3 1 -2 -3 -1 0
-1 -3 -2 -2 1 4 -1 -4 G 0 -2 0 -1 -3 -2 -2
6 -2 -4 -4 -2 -3 -3 -2 0 -2 -2 -3 -3 -1 -2 -1 -4
H -2 0 1 -1 -3 0 0 -2 8 -3 -3 -1 -2 -1 -2
-1 -2 -2 2 -3 0 0 -1 -4 I -1 -3 -3 -3 -1 -3
-3 -4 -3 4 2 -3 1 0 -3 -2 -1 -3 -1 3 -3 -3
-1 -4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 -2 2
0 -3 -2 -1 -2 -1 1 -4 -3 -1 -4 K -1 2 0 -1 -3
1 1 -2 -1 -3 -2 5 -1 -3 -1 0 -1 -3 -2 -2 0
1 -1 -4 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5
0 -2 -1 -1 -1 -1 1 -3 -1 -1 -4 F -2 -3 -3 -3
-2 -3 -3 -3 -1 0 0 -3 0 6 -4 -2 -2 1 3 -1
-3 -3 -1 -4 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3
-1 -2 -4 7 -1 -1 -4 -3 -2 -2 -1 -2 -4 S 1 -1
1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 1 -3 -2
-2 0 0 0 -4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1
-1 -1 -1 -2 -1 1 5 -2 -2 0 -1 -1 0 -4 W -3
-3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2
11 2 -3 -4 -3 -2 -4 Y -2 -2 -2 -3 -2 -1 -2 -3
2 -1 -1 -2 -1 3 -3 -2 -2 2 7 -1 -3 -2 -1 -4 V
0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2
0 -3 -1 4 -3 -2 -1 -4 B -2 -1 3 4 -3 0 1 -1
0 -3 -4 0 -3 -3 -2 0 -1 -4 -3 -3 4 1 -1 -4
Z -1 0 0 1 -3 3 4 -2 0 -3 -3 1 -1 -3 -1
0 -1 -3 -2 -2 1 4 -1 -4 X 0 -1 -1 -1 -2 -1 -1
-1 -1 -1 -1 -1 -1 -1 -2 0 0 -2 -1 -1 -1 -1 -1
-4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4
-4 -4 -4 -4 -4 -4 -4 -4 -4 1
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Expression

• A gene is expressed when the DNA sequence in the
  genome is transcribed into an mRNA molecule, and
  that mRNA molecule is correctly made into a
  protein (aka. string of amino acids for
  polypeptide).
• Note that evaluation of expression is often done
  by examining/counting the amount/number of mRNA
  molecules made by the cells of a particular
  tissue.

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DNA/RNA/Protein and Strands

• promoters
• anywhere from 1 to 10 KB to ??? upstream of a
  gene
• many proteins and other molecules (RNAs) involved
• largely unknown
• promoter bashing
• replace or delete regions of DNA in promoter
• measure level of expression
• trans- and cis- regulatory elements
• trans not co-localized to the gene
• cis generally localized to the gene

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Example -- LCR
An example of the functional potential for
non-coding regions is the locus control region of
the opsin gene cluster (Nathans, et. al. 1989)
shown to cause 50 of the cases of blue cone
monochromacy. The locus control region is
approximately 4 kilobases upstream of the red
opsin gene, and 43 kilobases upstream of the
green opsin gene. The 579 base region was mapped
to the X-chromosome using observed deletions
upstream of the red-green opsin gene cluster in
individuals with blue cone monochromacy. Blue
cone monochromatism is characterized by poor
central vision and color discrimination and
nearly normal retinal appearance.
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DNA/RNA/protein figure
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End
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Microarray Technology

• No genomics discussion would be complete without
  describing microarray technology.
• A powerful tool for genetic research which
  utilizes nucleic acid hybridization techniques
  and recent advancements in computing technology
  to evaluate the mRNA expression profile of
  thousands of gene in one single experiment.
• It has proven to be an extremely valuable method
  to better utilize the enormous amount of
  information provided by the completion of the
  human Genome Project.

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Gene Expression Motivation

• Pattern of gene expression in a cell is
  characteristic of its current state
• Virtually all differences in cell state or type
  can be correlated with differences in mRNA
  expression levels
• Expression patterns can provide clues to gene
  function and metabolic pathway architecture

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Potential Impact

• Preventative medicine
• Subtype diseases in order to design better drugs
  for a specific genotype
• More targeted drug treatment -- treat disease
  rather than symptoms

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Steps involved in Designing Microarray Experiment

• Preparation of fluorescently labeled target from
  RNA isolated from the biological sample (aka
  biological sample).
• Hybridization of the labeled target to the
  microarray.
• Washing, staining, and scanning of the array.
• Analysis of the scanned image.
• Generation of gene expression profiles.

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Physical Spotting
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DNA Array Technology
cDNA libraries and/or gene sequence data
Cell Lines
RNA
Hybridization
Surface
Target
Probe
Data Acquisition
Expression Levels
Analysis
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Probe Example
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(No Transcript)
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Microarrays What are they?
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Microarray Experiment
326 Rat Heart Genes, 2x spotting
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Affymetrix Technology
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Affymetrix Chip
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Hybridization/Microarray Tech.

• Very large scale
• multiples of 1K density for glass slides
• cheap
• custom
• considered not as reliable
• Affy U133
• 2 chips
• 45,000 probe sets
• 39,000 transcripts
• 33,000 genes
• SNP chip
• 11,500 SNPs (single nucleotide polymorphisms, or
  genotypes)
• 100,000 SNPs (another year?)
• Research and funding dilemma
• NIH sponsored funding
• only distilled data (if that) made available
• confidentiality issues

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Examples of Analysis

• simple filter
• all up, all down
• clustering
• Eisen diagrams
• volcano plots
• Mootha approach

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End Expression
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Polyadenylation (Poly-A)

• The addition of multiple adenines to a pre-mRNA
  and is part of the end of the transcription
  process
• Three steps
• 1) the RNA strand is cleaved at a particular site
• 2) the addition of poly-A's to the 3' end
• 3) the degradation of the remainder of the RNA
  transcript

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Polyadenylation
Cut
polyadenylated
degraded
AAAn
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Poly-A Signal

• AAUAAA specifies where the mRNA is cleaved, and
  the Poly-A is added
• Typically 23 or 24 bases downstream of this
  signal
• 10-200 A's added
• Increases translatability by about 20-fold
  (mechanism unknown).
• Also thought to improve stability protecting
  the end of the mRNA molecule from exonucleases.

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Alternative Polyadenylation

• common in human RNA (Edwards-Gilbert 1997)
• in many genes, 2 or more poly-A signals in 3 UTR
• alternative transcripts can show tissue
  specificity
• alternative poly-A signals may be brought into
  play following alternative splicing

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Edwards-Gilbert. Nucleic Acids Res, 13, 1997
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End