Transcript No Slide Title

Gene Expression
And Regulation
Bioinformatics
January 11, 2006
D. A. McClellan ([email protected])
Gene Expression
• Expressed in the transcriptome
• Every eukaryotic genome contains between
5000-60,000 protein-coding genes
• Only a small subset of those genes are
transcribed
Gene expression is regulated
in several basic ways
• by region (e.g. brain versus kidney)
• in development (e.g. fetal versus adult tissue)
• in dynamic response to environmental signals
(e.g. immediate-early response genes)
• in disease states
• by gene activity
Page 157
Central Dogma of Molecular Biology
DNA
RNA
protein
phenotype
cDNA
Page 159
DNA
RNA
protein
cDNA
DNA
RNA
protein
cDNA
UniGene
SAGE
microarray
Fig. 6.2
Page 159
Expression Databases & Analyses
• UniGene: for the comparison of cDNA
libraries
– Goals: (1) create one unique entry for each
gene, (2) collect all the ESTs associated with
each gene
• SAGE: Serial Analysis of Gene Expression
library
• DNA microarrays
5’ exon 1
3’
intron
exon 2
3’
exon 3 5’
intron
transcription
5’
3’
RNA splicing
(remove introns)
3’
5’
polyadenylation
5’
AAAAA 3’
Export to cytoplasm
Fig. 6.3
Page 161
Relationship of mRNA to genomic DNA for RBP4
Fig. 6.4
Page 162
Analysis of gene expression in cDNA libraries
A fundamental approach to studying gene expression
is through cDNA libraries.
• Isolate RNA (always from a specific
organism, region, and time point)
insert
• Convert RNA to complementary DNA
• Subclone into a vector
vector
• Sequence the cDNA inserts.
These are Expressed Sequence Tags
Page 162-163
UniGene: unique genes via ESTs
• Find UniGene at NCBI:
www.ncbi.nlm.nih.gov/UniGene
• UniGene clusters contain many ESTs
• UniGene data come from many cDNA libraries.
Thus, when you look up a gene in UniGene
you get information on its abundance
and its regional distribution.
Page 164
Cluster sizes in UniGene
This is a gene with
1 EST associated;
the cluster size is 1
Page 164
& Fig. 2.3,
Page 23
Cluster sizes in UniGene
This is a gene with
10 ESTs associated;
the cluster size is 10
Page 164
Cluster sizes in UniGene (human)
Cluster size
1
2
3-4
5-8
9-16
17-32
Number of clusters
10,400
7,100
6,800
5,300
3,800
3,100
500-1000
2000-4000
8000-16,000
16,000-30,000
UniGene build 186, 9/05
1,500
130
12
3
Page 164
Ten largest human UniGene clusters
Cluster
size
22,925
22,320
16,562
16,309
16,231
11,076
10,517
10,087
9,973
8,966
UniGene build 186, 9/05
Gene
eukary. translation EF (Hs. 522463)
eukary. translation EF (Hs. 4395522)
actin, gamma 1 (Hs.514581)
GAPDH (Hs.169476)
actin, beta (Hs.520640)
ribosomal prot. L3 (Hs.119598)
dehydrin (Hs.524390)
enolase 1 (alpha)(Hs.517145)
ferritin (Hs.433670)
metastasis associated (Hs.187199)
Table 6.2
Page 165
UniGene brain
libraries
UniGene lung
libraries
Fig. 6.7
Page 167
Brain Lung
Fig. 6.7
Page 167
CamKII
up-regulated
in brain
n-sec1 up-regulated
in brain
surfactant upregulated in lung
Page 167
Fisher’s exact test provides a p value
Digital differential display (DDD) results in UniGene
are assessed for significance using Fisher’s exact test
to generate a p value.
p=
NA! NB! c! C!
(NA + NB)! g1A! g1B! (NA – g1A)!(NB – g1B)!
The null hypothesis (that gene 1 is not differentially
regulated in a comparison of two libraries) is rejected
when p is < 0.05/G (where G = the number of UniGene
clusters analyzed).
Pages 165
Pitfalls in interpreting cDNA library data
• bias in library construction
• variable depth of sequencing
• library normalization
• error rate in sequencing
• contamination (chimeric sequences)
Pages 166-168
http://mgc.nci.nih.gov
Fig. 6.8
p. 168-169
Serial analysis of gene expression (SAGE)
• 9 to 11 base “tags” correspond to genes
• measure of gene expression in different
biological samples
• SAGE tags can be compared electronically
Page 169
SAGE tags are mapped to UniGene clusters
Tag 1
Tag 1
Tag 2
Tag n
Cluster 1
Cluster 2
Cluster 3
Cluster 1
Page 169