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).

Genome... the dynamic complement of heritable genetic material, Transcriptome... mRNA component in an individual, Proteome... the protein component of an individual, Expanded Central Dogma GenomeTranscriptomeProteome

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.

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 appropriate components on this figure. Please study this slide.

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. The Experiment Part I: Transcriptome Analysis

Affymetrix probe U95A array... 63,000 probe set 10,000 Human genes 5 arrays Affymetrix DNA Chip Part I: Plan A

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? Changes in gene expression are greatest in the Human brain gene cluster. Primates Mice

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.

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. Part I: Plan B

Microarrays...denatured, double stranded DNA ( 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.

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. The Experiment Part II: Proteome Analysis

Proteomics (2d-gels) Proteins separated by mass, then by charge. Qualitative (positions), Quantitative (amount), Present/absent (+/-) Human Chimp

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?

Sugar and Genetics –PDF of paper available on WEB page, print pp Monday take home quiz due

Dead Week Monday: Sugar Paper, Wednesday: Pheromone paper, Friday: review, Take home exam due midnight Weds. Office Hours: as usual.