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Biochemistry 412
Overview of Genomics & Proteomics
(con’d)
21 January 2005
After the human genome and the genomes of the model
organisms S. cerevisiae, C. elegans, D. melanogaster, and
M. musculus, what is left to sequence??
Other animal genome sequences also in progress:
Cow
Chicken
Polyp hydra
Dog
Clawed frog
Starlet sea anemone
Coyote
Mosquito (several)
Acorn worm
Wolf
Honey bee
Blood fluke
Guinea pig
Silkworm
Flat worm
Nine-banded armadillo Multiple fruit fly species Surf clam
Cat
Tobacco budworm
Rat
Lemur
Red flour beetle
Orangutan
Elephant
Zebrafish
Baboon
Rhesus monkey
Pufferfish
Chimpanzee
Wallaby
Freshwater snail
Duck-billed platypus
Opposum
Sea squirt
Rabbit
Note: plus literally hundreds of microbial and other lower organism genomes!
The human genome sequence is finished….
>>> But what other genome-based studies
have been enabled by this achievement?
Some examples:
• Human variation and evolution (e. g., “SNPs”)
• Somatic mutations (e. g., loss-of-heterozygosity in cancer)
• RNA expression profiling (cf. “DNA chips”)
• Methylation patterns (e. g., epigenetics and gene silencing)
Single Nucleotide Polymorphisms
(“SNPs”)
Roses (2000) Nature 405, 857.
Microarrays
(DNA Chips)
Note: 4N masks required to
make an array of oligonucleotides
each of length N.
Pease et al (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5022.
Note: this is the photolabile blocking
group, “X”, indicated schematically
in Figure 1.
Pease et al (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5022.
Key feature: known oligo
sequence at each “address”
on the chip.
Lipshutz et al (1999) Nature Genet. (suppl.) 21, 20.
Lipshutz et al (1999) Nature Genet. (suppl.) 21, 20.
RNA Profiling
Lockhart & Winzeler (2000) Nature 405, 827.
a) In situ synthesis of oligonucleotides on slide
b) Post-synthesis spotting of cDNAs on slide
mRNA Expression Profiling with Oligonucleotide Microarrays
(Affymetrix chip)
…some tricks of the technique
Lipshutz et al (1999) Nature Genet. (suppl.) 21, 20.
Lockhart & Winzeler (2000) Nature 405, 827.
Note that the technique is sensitive enough to
detect as little as one mRNA molecule per cell!
Highly parallel genomics-based analysis methods
are ideal for identifying cross-correlations (e.g.,
co-expression, co-regulation, sharing of binding
partners, etc.) and other associations among
disparate genes or gene products. Clustering
algorithms can be used to highlight these
relationships. The desire to understand the
biological relevance of such data has been one of
the strong driving forces behind the development
of the field of systems biology.
An example from yeast
Bassett et al (1999) Nature Genet. (suppl.) 21, 51.
Note: caloric restriction gene chip experiment w/ rats.
Ref: Lee et al (1999) Science 285, 1390.
Lee et al (1999) Science 285, 1390.
DNA Chips and Drug Discovery
Pharmaceutical companies also use mRNA profiling methods to
search for new drug targets. For example, one might compare
mRNA extracted from normal vs. cancerous liver tissue and look
for genes that are up-regulated in the tumor. If such genes code
for proteins that are necessary for maintenance of the tumor but
are less important to normal, slowly-dividing cells, then these
proteins may be candidates for novel drug discovery and
development programs. Examples of such genes are those
involved in vascularization of the tumor, maintenance of telomeres,
etc. [Note: some genes identified in this way may have biochemical
functions that are as yet unknown, which has motivated the field of
functional genomics.]
Kapranov et al (2002) Science 296, 916.
Surprise: not all (or even most) transcription comes
from protein coding regions of the genome!
“Proteomics”
The study of the complete complement
of proteins found in an organism
“Degrees of Freedom” for Protein Variability
Covalent Modifications in Proteins
• Post-translational modifications (e.g., phosphorylation, glycosylation, etc.)
- more than 200 such modifications are known, and
they can occur at multiple sites in a single protein
• Alternative splicing of a primary transcript
- in extreme cases, a single gene can produce
tens of thousands of different mRNAs!
• Proteolytic processing
• Protein aging
Thus, there are probably many millions
of different proteins in our bodies!!
More Reality Therapy re Proteins
• They have “personalities”: each behaves differently
• They exist in different concentrations, ranging over a
million-fold
• It will be extremely difficult to even identify them all
(see previous slide)
Take-home message:
Proteomics presents challenges that are
orders-of-magnitude more difficult
than those presented by genomics!
“Classic” Proteomics: 2-Dimensional Gel Electrophoresis
<--------- separation by size ----------->
<-------- separation by charge -------->
Pandey & Mann (2000) Nature 405, 837.
Mass spectrometry is a major tool in proteomics.
Comparative Protein Profiling Using 2D Gels and Mass Spectrometry
Pandey & Mann (2000) Nature 405, 837.
Note: sometimes looking at the proteins gives a different
impression than one gets from looking at the mRNAs!
Protein chips
Pandey & Mann (2000) Nature 405, 837.
Yeast Two-Hybrid System (Song and Fields)
Pandey & Mann (2000) Nature 405, 837.