Transcript How are we different? …at the RNA level.

Exam #2
Mean = 73%
Median = 74%
Mode = 90%
A range: | | | | | | | | |
B range: | | | | | | | | |
C range: | | | | | | |
D range: | | | | | | | | | |
Failing: | | | | | | | | |
For Final:
Final - Exam #2
2
- or Final - Final Average
2
…whichever is higher.
How are we different?
…at the RNA level.
Northern Analysis
DNA hybridizing to RNA,
DNA Arrays and Expression
…grid gene-specific ASOs onto the DNA
chip, or “known” cDNAs onto microarrays,
– extract mRNA from a specific tissue,
– make fresh cDNA from the new mRNA,
• and label it,
– bind to the array for display.
http://www.bio.davidson.edu/courses/genomics/chip/chip.html
Genes and Targets
• With many genome projects finished done, most, if not all
of the genes identified can be gridded,
– presently, several completely sequenced genomes have been
gridded,
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H. sapiens (>20,000 genes)
Arabidopsis (>26,000 genes),
C. elegans (>22,500 genes),
Drosophila (>13,500 genes),
yeast (>6000 genes),
more,
• drug identification, fundamental research, etc.
www.affymetrix.com
Gene/Drug Discovery
…genes involved in cancer and other diseases have
been identified through a variety of techniques,
– genome expression analysis provides a means of
discovering other genes that are concomitantly
expressed,
– genome expression analysis provides a means of
monitoring drug/treatment regimes.
Applications
• Can study the role of more than 1700 cancer
related genes in association with the (rest) of the
genome,
• Define interactions and describe pathways,
• Measure drug response,
• Build databases for use in molecular tumor
classifications,
– benign vs. cancerous, slow vs. aggressive.
Extended Applications
• Water quality testing (4 hours vs. 4 days),
• Environmental watchdogs,
• Fundamental research on non-human subjects,
• Direct sequencing of related species for evolutionary
studies,
• Comparisons of gene regulation between closely related
species,
• etc.
Monday
•Human and chimp DNA is ~98.7
similar,
•But, we differ in many and
profound ways,
•Can this difference be attributed,
at least in part, to differences in
gene expression, rather than
differences in the actual gene and
gene products?
How are we different?
…at the RNA level.
…and at the protein level.
Gene Expression Technologies
• General Scheme: Extract mRNA, synthesize labeled cDNA,
Hybridize with DNA on the array,
– DNA Chips (Affymetrix) and MicroArrays can measure mRNA
concentration of thousands of genes simultaneously,
– look for genes that are expressed similarly (clustering).
What is clustering?
Linear
Round
Cluster Analysis Yields Dendrogram
T (RESOLUTION)
What’s the Question
• Human and chimp DNA is ~98.7
similar,
• But, we differ in many and
profound ways,
– can this difference be attributed, at
least in part, to differences in gene
expression, rather than differences
in the actual genes and gene
products?
Especially in the brain.
Huh?
• Prevailing notion (though fading fast):
– a structural gene is mutated,
– alleles that code for favorable adaptive proteins
survive,
– and, in fact, out-compete old alleles…evolution
marches on.
• Paper’s hypothesis: it’s not just the genes that are
changing, but the REGULATION of the genes,
– cis and trans acting factors are considered here.
Why Brain?
Why Regulation?
• The human genome contains about 20k - 25k
genes,
– Drosophila, 13,061,
– Arabidopsis, 26,027,
– C. elegans, 19,099 genes.
• One hypothesis: “many” of the additional genes
found in complex organisms, are transcription
factors,
• more complex control of transcription leads to more
complex organisms.
First
...What does it mean that our genomes are
98.7% similar at the DNA level, and how do
we know this?
Human/Chimp BES Similarity
BAC End Sequences
This represents coding and non-coding regions of the genome.
Are our Phenotypes 98.7% Similar?
• Some apparent differences,
– HIV susceptibility, epithelial neoplasms (cancers),
malaria, and Alzheimers,
• In fact, there is only one well understood
biochemical difference,
– A 92 bp deletion in a gene that codes for a hydroxylase,
results in an un-hydroxylated secretion protein in our
immune system.
The Experiment
Part I: Transcriptome Analysis
• Check patterns of gene expression
level,
– using DNA Chip arrays,
– for >10,000 genes in humans, chimps,
orangutans, and macaques,
• Compare brain, liver, and blood
specifically,
•
Perform cluster analysis and compare
the amount of change in expression
levels across the genome.
T (RESOLUTION)
Part I: Plan A
Affymetrix
probe
• U95A array...
63,000 probe set
10,000 Human genes
5 arrays
Affymetrix DNA Chip
Targets
• Labeled Human cDNA,
Chimp cDNA, Macaque
cDNA,
– collect tissue (from cadavers),
– brain, blood, liver,
– extract RNA, make cDNA
– label cDNA.
Cluster Analysis
Distances represent the relative differences in gene
expression changes.
So What?
Primates
Mice
• Changes in gene expression are greatest in the
Human brain gene cluster.
Probably Rejected by the Journal
• Why?
– Probe was human, target chimp and ape mRNA
sequence may slightly differ,
• at the “allele specific oligonucleotide” level, single
base changes may skew the data.
Part I: Plan B
Microarray
better probe
• Spotted 17,997 PCR products
onto nylon, probed with labeled
cDNAs,
– PCR primers are available, in kits,
that will amplify just about any
part of the human genome,
– 1,000 bp fragments were
generated,
• Base pair differences won’t affect
probe sensitivity over this large a
target.
Microarrays
...denatured, double stranded DNA (500 - 5000 bp) is dotted,
or sprayed on a glass or nylon substrate,
...up to tens of thousands of spots per array,
quill technology...
Microarray Data
17,997 genes
5:1 difference in
expression profiles.
The Experiment
Part II: Proteome Analysis
• Check for differences in protein
expression using 2-D gel analysis,
– differences in location on the gel
indicate qualitative differences,
– differences in amount indicate
quantitative differences,
• Controls,
– Rodent tests.
Proteomics
(2d-gels)
Human
Chimp
• Proteins separated by mass, then by charge.
• Qualitative (positions), Quantitative (amount), Present/absent (+/-)
8500 Protein Spots
What do You Think?
Microarray Data
1b
What does this figure
demonstrate?
How is the data
different from Fig.
1a?
Proteomics
(2d-gels)
• What does this figure show?
• What conclussions were drawn from the data?
• Does it support the microarray data?
Final Review
• Friday: Pheromone paper.
• Friday December 7th. Lecture time
will be used for review,
• Final Tuesday 10:30 - 12:30,
• Office Hours: as usual.