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Life is Dynamic

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Title: Life is Dynamic


1
Life is Dynamic
2
The information
Regulated by proteins
3
Autosomes
  • gt 22 matched sets (44 total)
  • gt 100 identical in males and females
  • All sequences for a particular mRNA are not
    identical
  • Some sequences are defective, some are just a
    little different

4
Males and Females
  • Autosomes 22 matched set
  • Sex chromosomes the difference

5
Chromosome Squashes
Human Chromosome Launchpad
http//www.ornl.gov/hgmis/launchpad/
NCBI
http//www.ncbi.nlm.nih.gov/
http//www.ncbi.nlm.nih.gov/LocusLink/list.cgi
6

Genetics and Translation Gene Expression
  • There are 4 different RNA nucleotides that can
    pair with the 4 different DNA nucleotides
  • Messanger RNA (mRNA) is a small sequence of RNA
    nucleotides that describes the DNA code for the
    protein.
  • The rest of the small DNA sequences in the
    incredibly large DNA molecule we call a
    chromosome is WHEN to make the molecule.

7
Proteins
  • Coded by DNA
  • Every three nucleotides in a gene sequence code
    designates the code for one amino acid specific
    type of amino acid in a protein sequence
  • A protein is a sequence of amino acids
  • To Make a protein, a cell must transcribe (Copy)
    the DNA coding sequence into a portable message
    (mRNA) that can leave the nucleus of the cell and
    be used to make the protein.

8
Transcription DNA mRNA
Nucleus
Translation mRNA Protein
Cytoplasm
9
Proteins are made of Amino acids
10
Regulation of Gene Expression
Transcription factors
11
Measuring gene expression (mRNA)
1.Coding sequences are not entirely unique 2.
Motifs 3. Hybridization 4. http//www.ncbi.nlm.nih
.gov80/blast
12
Gene Expression
13
Other Patterns
14
Gene Arrays
Two basic types 1. Spotted macro-arrays 2.
Synthesized micro-arrays
15
Spotted macro array
16
Results Liver, Control vs treated (5.5hr.)
51 identifiable genes Genes Involved in Acute
phase/Immune Response Genes Involved in Energy
Metabolism Genes Affecting Microsomal Enzymes
Alterations of Other Genes eg protein synthesis,
signal transduction
17
Genes Involved in Acute phase/Immune Response
? Pre-pro-complement C
host immune response (22, 55) ?
Parathymosin
thymocyte maturation (56, 57) ? MHC class 1
presages immune-mediated
damage (58-60) ? 2, 5, - OAS
INF-induced antiviral response (23,
61) ? target of antiproliferative antibody
regulation of mitotic activity (62,
63) ? anti-acetylcholine receptor antibody
immunoglobulin expression ?
thiol-specific antioxidant
NF?B signaling pathway (64, 65) ? Biglycan
increased in
inflammation (66-68) ? ?2 - ? globulin

acute phase protein (69) ? growth hormone
regulated protein
anti-protease (70, 71) ? thrombin
blood
clotting/pro-inflammatory (24, 72) ? ?1 -
macroglobulin inhibits
inflammatory proteases (73) ? ?2 - macroglobulin
inhibits inflammatory proteases
(25) ? ?1 - acid glycoprotein
inhibits microvascular permeability (26, 27,
74) ? serum amyloid P
scavenges chromatin (75, 29) ? Vitamin D
binding protein Vitamin D
carrier/scavenges actin (28, 76) ? ? -fibrinogen
blood clotting (30) ?
fibrinogen ?-chain blood
clotting (30) ? fibrinogen ?-chain
blood clotting (30) ? proline rich
protein blood clotting (77) ?
metallothionein-2/-1 metal
binding protein (32, 78) ? metallothionein-I
metal binding protein (32,
78) ? delta-aminolaevulinate synthase
heme synthesis (31, 79)
18
Experimental Design
  • Rats were sacrificed at 17 time points after MPL
    administration
  • (between 0 and 72 hours)
  • 4 controls
  • 3 rats per time point

50 mg/kg dose of methylprednisolone sodium
succinate (MPL) in the right external jugular vein
Liver and muscle flash frozen
Trizol
Eg. one hour
Total RNA
Total RNA
biotin-labeled cRNA
hybridize
cDNA
Each chip contains cRNA from one rat
Total RNA
biotin-labeled cRNA
cDNA
hybridize
Total RNA
hybridize
biotin-labeled cRNA
cDNA
19
(No Transcript)
20
Trees of Normalized Liver and muscle
21
Filtering and Clustering
The goal is to separate the regulated genes
from the unregulated genes. Start with
filtering to reduce the number of genes. Reducing
the number of genes makes statistical approaches
more powerful
22
Muscle Eliminate genes not present on at least 4
chips
23
Filter on response characteristics
0.65 two conditions
1.5 two conditions
24
ANOVA Bonferroni post test
25
Recombine lists for clustering
26
A Few Genes
C/EBP
MyoD
Glutamine Synthetase
27
A Few More Genes
Mt1
EST AA859663
Cyclin D1
28
Mechanism of Action for Glucocorticoids (GC)
EFFECTS (Direct)
EFFECTS (Direct)
EFFECTS (Direct)
EFFECTS (Direct)
protein binding
protein binding
diffusion
diffusion
Cytosol
Cytosol
Cytosol
Cytosol
P
binding
P
binding
activation
90
90
56
56
activation
90
90
70
70
P
P
90
90
56
56
translocation
translocation
90
90
70
70
dimerization
dimerization
  • Mediated by Glucocorticoid Receptor
  • Acute effects
  • (e.g. adrenal suppression,
  • lymphocyte trafficking)
  • Gene-mediated effects
  • via GRE
  • (e.g. TAT induction,
  • PEPCK induction)

Nucleus
Nucleus
Nucleus
Nucleus
P
P
P
P
P
P
P
P
P
P
P
P
Target DNA
Target DNA
Target DNA
Target DNA
P
P
P
P
GRE
GRE
GRE
GRE
transcription
transcription
Repressed mRNA
Induced mRNA
Repressed mRNA
Induced mRNA
Repressed mRNA
Induced mRNA
Repressed mRNA
Induced mRNA
translocation
translocation
Induced mRNA
Repressed mRNA
Induced mRNA
Repressed mRNA
Induced mRNA
Repressed mRNA
Induced mRNA
Repressed mRNA
translation
translation
Induced Protein
Induced Protein
Repressed Protein
Repressed Protein
Induced Protein
Induced Protein
Repressed Protein
Repressed Protein
EFFECTS (Gene
-
Mediated)
EFFECTS (Gene
-
Mediated)
EFFECTS (Gene
-
Mediated)
EFFECTS (Gene
-
Mediated)
29
Methylprednisolone PK/PD in Rats
Hepatic Cytosol GR Density
MPL Plasma Conc (ng/ml)
Nuclear Receptors Binding DR (N)
(fmole/mg protein)
Time, hour
TAT Activity (unit/mg protein)
TAT mRNA (pmol/g liver)
Time, hour
30
Cluster 1 (23 probes, 21 genes)
Cluster 3 (21 probes, 18 genes)
Cluster 2 (9 probes, 6 genes)
Cluster 6 (67 probes, 44 genes)
Cluster 5 (8 probes, 5 genes)
Cluster 4 (66 probes, 49 genes)
After cluster analysis using GeneSpring?
31
Gene Array (cluster1 ? TAT Like mRNAs)linear
stimulation by DR(N) on mRNA production
I(t)Rm
mRNAR
kd_Rm
ks_Rm
ks_R
DR
S
mRNA
kon
kT
ks_m
D R
DR(N)
kd_m
Rf kre
kd_R
(1-Rf) kre
32
Mechanistic Modeling of Cluster 1 and 2
Cluster 1
Cluster 2
33
Inhibition of Expression
34
Inhibition Followed by Stimulation
35
Fit of Data to Previous Set of Equations
             
 
36
Pathway Modeling
Kyoto Encyclopedia of Genes and Genomes
http//www. genome.ad.jp/ kegg/metabolism.html
International Union of Biochemistry and Molecular
Biology
http//www.chem.qmul.ac.uk/ iubmb/enzyme/
37
Transcription Factor Modeling
38
Model for C/EBP Induction of Arginase
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