1

Basic Biology Related to Technology for Measuring

Gene Expression

1/11/2011

Copyright © 2011 Dan Nettleton

2

Central Dogma

DNA

RNA

protein

(transcription)

(translation)

Proteins perform essential biological functions.

DNA contains genes that code for proteins.

3

A VERY Simplified Eukaryotic Cell

nucleus

chromosome

DNA strands

DNA contains thousands of genes.

cytoplasm

4

Deoxyribonucleic Acid (DNA)P

S

P

S

P

S

P

S

P

S

P

S

P

S

P

S S S S S S S

P P P P P P P P

5’ 3’

5’3’

A A T T G C C

T T A A C G G

S=deoxyribose sugarP=phosphate

BasesA=adenineC=cytosineG=guanineT=thymine

hydrogenbonds

The two DNA strands are twisted inthe famous double helix shape (basepairs like rungs in a spiraling ladder).

......

... ...

purinespyrimidines

5

Nucleotide=Phosphate-Sugar-BaseP

S

P

S

P

S

P

S

P

S

P

S

P

S

P

S S S S S S S

P P P P P P P P

5’ 3’

5’3’

A A T T G C C

T T A A C G G

......

... ...

6

Sequence of bases holds instructions for manufacturing proteins

P

S

P

S

P

S

P

S

P

S

P

S

P

S

P

S S S S S S S

P P P P P P P P

5’ 3’

5’3’

A A T T G C C

T T A A C G G

......

... ...

CodingSequence

7

RNA Polymerase

• An enzyme is a protein that catalyzes chemical reactions.

• RNA polymerase is the enzyme responsible for transcribing DNA to RNA.

8

Transcription

P

S

T

P

S

A

P

S

A

P

S

C

P

S

G

P

S

P

3’

G

...P

S5’

T

...

S S S S S S S

P P P P P P P P

5’3’

A A T T G C C

... ...

9

Transcription

P

S

A

P

S

A

P

S

C

P

S

G

P

S

U

P

S

P

3’

G

...P

S5’

U

...

S

P

A

S

P

T

S

P

T

S

P

G

S

P

C

S

P

3’

A

... S

P P

5’

C

...

10

Ribonucleic Acid (RNA)

P

S

A

P

S

A

P

S

C

P

S

G

P

S

U

P

S

P

3’

G

...P

S5’

U

...

BasesA=adenineC=cytosineG=guanineU=uracil

S=ribose sugarP=phosphate

The product of transcriptionis known as the

primary transcript.

11

Posttranscriptional Modificationsto Primary Transcript

Primary transcript

Intervening sequences corresponding to intronsthat are removed through splicing

3’ UTR5’ UTR

Primary transcript after modification: messenger RNA (mRNA)

AAAAAA...AAAA

poly-A tailCoding portions of RNA sequence

corresponding to exons

5’ UTR 3’ UTR

5’ cap

G

12

Expression Levels

• The amount of messenger RNA (mRNA) produced by a gene is known as the gene's expression level.

• This course focuses on the design and analysis of experiments that involve the simultaneous measurement of gene expression levels for thousands of genes.

13

Transcription takes place inside the nucleus.

nucleus

chromosome

DNA strands cytoplasm

Translation takes place outside the nucleus.

14

Translation

mRNA

Ribosome

amino acid sequence

folds to become a protein

15

During translation transfer RNA (tRNA) translates the genetic code

... ...A A C GU GU

codon codon

A A U

leu

U G C

thr

tRNAanticodon

amino acids

16

The Genetic Code

UUU phe UCU ser UAU tyr UGU cysUUC phe UCC ser UAC tyr UGC cysUUA leu UCA ser UAA STOP UGA STOPUUG leu UCG ser UAG STOP UGG trp

CUU leu CCU pro CAU his CGU argCUC leu CCC pro CAC his CGC argCUA leu CCA pro CAA gln CGA argCUG leu CCG pro CAG gln CGG arg

AUU ile ACU thr AAU asn AGU serAUC ile ACC thr AAC asn AGC serAUA ile ACA thr AAA lys AGA argAUG met ACG thr AAG lys AGG arg

GUU val GCU ala GAU asp GGU glyGUC val GCC ala GAC asp GGC glyGUA val GCA ala GAA glu GGA glyGUG val GCG ala GAG glu GGG gly

Firs

t B

ase

Second Base

U

C

A

G

U C A G

mRNAcodon

aminoacid

17

Miscellaneous Comments

• The biology is more complicated than I described.

• It is amazing!

• Humans have somewhere around 30,000 genes. (The exact number is a subject for debate.)

• Much of the variation is created by differences in how cells use the genes they have.

• Microarrays and Next Generation Sequencing are tools that can help us understand how cells of various types use their genes in response to varying conditions. This helps us to understand gene function.