Human Monday
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).
Expanded Central Dogma
Genome
Transcriptome
Proteome
Genome... the dynamic complement of heritable genetic material, Transcriptome... mRNA component in an individual, Proteome... the protein component of an individual,
Whats 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 allelesevolution marches on.
Papers hypothesis: its not just the genes that are changing, but the REGULATION of the genes,
cis and trans acting factors are considered here.
Are our Phenotypes 98.7% Similar?
Some apparent differences,
HIV susceptibility, epithelial neoplasms (cancers), malaria, and Alzheimers,
In fact (in 2002), there was 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.
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.
Chimp Genomic Sequence, 2005
GO, Gene Ontology
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.
Eukaryotic Transcription Initiation
Apply the terms from the previous slide to the components appropriate on this figure.
Please study this slide.
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 comparative analysis and compare the amount of change in expression levels across the genome.
Part I: Plan A
Affymetrix
probe
U95A array...
Affymetrix DNA Chip
63,000 probe set 10,000 Human genes 5 arrays
Targets
Labeled Human cDNA, Chimp cDNA, Macaque cDNA,
collect tissue (from cadavers), brain, blood, liver, extract RNA, make cDNA label cDNA.
Difference 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 wont 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...