chapter 22— genomics ii functional genomics—studying genes in groups, with respect to the cell,...

Chapter 22—Genomics II

• Functional Genomics—studying genes in groups, with respect to the cell, tissue, signaling pathway or organism

• Proteomics—to understand the interplay among many different proteins (cellular processes and organismal level [traits])

• Bioinformatics—using computers, math, and statistics to understand the genome and proteome information (record, store, analyze, predict)

Add reverse transcriptase, poly-dTprimers that anneal to the mRNAs,and fluorescent nucleotides.Note: Only 1 complementarycDNA strand is made.

View with a laser scanner.

Hybridize cDNAsto the microarray.

A mixture of 3different types ofmRNA

A portion of a DNA microarray

Fluorescentlylabeled cDNA thatis complementaryto the mRNA

A

A

AA B

C D

E F

A B

C D

E F

D

FF

F

D

D

A

AA

D

FF

F

D

D

Figure 22.1 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Microarrays for studying gene

expression or re-sequencing

Modern day “Southerns” and “Northerns”—microarray analysis

Two distinct forms of large B-cell lymphoma are shown by the expression pattern: GC B-like DLBCL (orange) and Activated B-like DLBCL (blue)

ASH ALIZADEH et al. 2000Nature 403, 503-511 (3 February 2000)

significantly better overall survival

Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling

Observation/problem• Diffuse large B-cell lymphoma (DLBCL) = most common subtype of non-Hodgkin's

lymphoma is clinically heterogeneous: 40% of patients respond well to current therapy and have prolonged survival, whereas the remainder succumb to the disease

Hypothesis• variability in natural history reflects unrecognized molecular heterogeneity in the

tumours.

Experiment• DNA microarrays used for a systematic characterization of gene expression in B-cell

malignancies.

Results• Diversity in gene expression among the tumours of DLBCL patients (reflecting the

variation in tumour proliferation rate, host response and differentiation state of the tumour).

• Identified two molecularly distinct forms of DLBCL which had gene expression patterns indicative of different stages of B-cell differentiation.

– One type expressed genes characteristic of germinal centre B cells ('germinal centre B-like DLBCL'); – the second type expressed genes normally induced during in vitro activation of peripheral blood B

cells ('activated B-like DLBCL').

• Patients with germinal centre B-like DLBCL had a significantly better overall survival than those with activated B-like DLBCL.

Conclusion• Molecular classification of tumours on the basis of gene expression can thus identify

previously undetected and clinically significant subtypes of cancer.ASH ALIZADEH et al. 2000

Nature 403, 503-511 (3 February 2000)

Add formaldehyde to crosslinkprotein to DNA. Lyse the cells.Sonicate DNA into small pieces.

Add antibodies that recognize theprotein of interest. The antibodiesare bound to heavy beads. Afterthe antibodies bind to the proteinof interest, the sample issubjected to centrifugation.

Collect complexes in pellet.Add chemical that breaks thecrosslinks to remove the protein.

Unknown Candidates:Ligate DNA linkers to theends of the DNA.

Known Candidates: Conduct PCR using primersto a known DNA region.

If PCR amplifies the DNA,the protein was bound tothe DNA region recognizedby the primers.

Conduct PCR using primersthat are complementary tothe linkers. Incorporatefluorescently labelednucleotides during PCR.

Denature DNA andhybridize to a microarray.

Antibody againstprotein of interest

Protein of interest

Bead

Protein of interest

Linker

or

Pellet

See Figure 22.1

Figure 22.2

Which DNA sequences bind to my protein of

interest?

Chromatin Immunoprecipitation Assay (ChIP)

Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6

Exon 1Exon 2

Exon 4Exon 5

Alternative splicingTranslation

Exon 6

Exon 1

(a) Alternative splicing

Exon 3Exon 4

or

or

Exon 5Exon 6

pre-mRNA

Exon 1Exon 2

Exon 4Exon 6

Why is the proteome so large? Alternative splicing

Proteolyticprocessing

Attachment ofprostheticgroups, sugars,or lipids

Sugar

Hemegroup

Phospholipid

Disulfide bondformation

S SSH SH

Irreversible modifications

(b) Posttranslational covalent modification

Phosphorylation

Methylation

Phosphategroup

Acetylgroup

Methylgroup

PO42-

C

CH3

CH3

O

Reversible modifications

Acetylation

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Why is the proteome so

large? Post translational

modification

SDS-polyacrylamide gel

Proteins migrate until theyreach the pH where theirnet charge is 0. At thispoint, a single band couldcontain 2 or moredifferent proteins.

Lyse a sample of cells andload the resulting mixtureof proteins onto an isoelectricfocusing gel.

pH 10.0pH 4.0

pH 10.0

pH 4.0

200 kDa

10 kDa

Lay the tube gel onto anSDS-polyacrylamide gel andseparate proteins accordingto their molecular mass.

Techniques to study the

proteome: 2D Gel analysis

Brooker, Fig 22.4

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Digest protein intosmall fragmentsusing a protease.

Determine the massof these fragments witha first spectrometer.

C

N

C

N

Purified protein

Mass/charge

Ab

un

dan

ce

0 4000

1652 daltons

Techniques to study the proteome:

Mass spectrometry

Brooker, Fig 22.5

Brooker, Fig 22.5, cont.


Analyze this fragment witha second spectrometer.The peptide is fragmentedfrom one end.

Mass/charge

Ab

un

dan

ce

0 4000

1652 daltons

Mass/charge

Ab

un

dan

ce

900

–Asn–Ser–Asn–Leu–His–Ser–

10081114

12011315

1428

1565

1652

1800

Tandem mass spectrometry to

sequence peptides

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or displayNumbers represent the base number

in the sequence file

Example of DNA Sequence as stored in Genetic Database

A bioinformatics program may ask:• Does the sequence contain a gene?• Which nt’s are the functional sites (e.g. promoters,

exons, introns, termination sequence)?• Does the sequence encode a protein? (have an open

reading frame [ORF]• What is the secondary structure of its RNA or

associated amino acid sequence?• Is the sequence homologous to any other known

sequences?• What is the evolutionary relationship between two

or more sequences?

3′ end

5′ end


Brooker, Fig 22.7

A secondary structural model forE. coli 16S rRNA


• DNA sequences of the lacY gene– ~ 78% of the bases are a perfect match

• In this case, the two sequences are similar because the genes are homologous to each other– They have been derived from the same ancestral gene– Refer to Figure 22.6

Sequence matches between E. coli and K. pneumoniae

Human Pa Ca

Mouse Lu Ca

Human LHON, Human Thy Ca

Mouse Lu Ca

Example output from a computer alignment program (and

comparison to real world data)

Interesting cancer mutation pattern in mitochondrial ND6 protein

Sequence homology used to “hang” human cancer mutations on the bovine crystal structure of Cytochrome B

Chen and Uberto 2014

Federal Genetic Databases

National Center for Biotechnology Informationwww.ncbi.nlm.nih.gov/

U.S. government-funded national resource for molecular biology information.

http://www.ncbi.nlm.nih.gov/

BLAST programs identify sequences with homology or similarity

Table 22.5


Figure 22.6

Accumulation ofrandom mutationsin the 2 genes

Mutation

Ancestral lacY gene

Ancestralorganism

Evolutionary separationof 2 (or more)distinct species

lacY gene

E. coli

lacY gene

lacY gene lacY gene

K. pneumoniae

Mutation

Origin of orthologous

genes

Myoglobin

a chains b chains

Hemoglobins

Mil

lio

ns

of

year

s ag

o

1,000

800

600

400

200

0

Mb ζ ψζ ψα2ψα1 α2 α1

f ε gG gA ψβ δ β


Brooker, Fig 8.7

DuplicationBetter at binding and

storing oxygen in muscle cells

Better at binding and transporting oxygen via red

blood cellsAncestral globin

Orthologs, paralogs, homologs

From Thompson and Thompson,

Genetics in Medicine, 6th ed.

Like Brooker fig 8-7

• All the globin genes have homology to each other• a-like genes are paralogs of each other; • b-like genes are paralogs of each other; • a-1 in mice and a-1 in humans are orthologs

chapter 22— genomics ii functional genomics—studying genes in groups, with respect to the cell,...

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