lecture 3 - genomes and transcription

Fig. 6-1

What is in our genome?

Ge

ne

s

A large fraction of our genome does not produce

gene products (with known function)!

Simple-sequence - repetitious DNA

(≈6% of human genome)

Satellite DNA: 14-500 bp repeats in tandem, 20-

100kb (Heterochromatin – telomeres and

centromeres)

Microsatellites: 1-13bp repeats in tandem, up to

150bp

How do these

repeats arise? Fig 6-39

Figure 21.7

1. Homologs pair up.

2. Repeats misalign.

Crossing over and

recombination occur.

3. New repeat

numbers are created.

8 repeats

8 repeats

10 repeats

6 repeats

Chromosomes break

and exchange here

The number of repeats can change through unequal

crossing over during meiosis

Result: # repeats can be highly

variable between individuals - useful for DNA fingerprinting or

disease diagnosis Biology (Campbell, 9 ed) Fig 13-12

Meiosis I:

Meiosis II:

n - 1

Simple-sequence repeats may occur through

replication errors

Fig 6-5

The PCR method is used to amplify DNA sequences

– Polymerase chain reaction (PCR) is a method of amplifying a specific segment of a DNA molecule

– Relies upon a pair of primers – Short DNA molecules that bind to sequences at each end

of the sequence to be copied

– Used as a starting point for DNA replication

Copyright © 2009 Pearson Education, Inc.

//localhost/../../Program Files/TurningPoint/2003/Questions.html

Fig 5-20

heat, 95˚C

cool, 50-72˚C

72˚C

DNA template

Primer Primer

5’

5’ 3’

3’ 3’ 5’ 5’ 3’

Basics of PCR (polymerase chain reaction)

WHAT IS NEEDED FOR A PCR REACTION?

Exponential amplification of DNA – up to 2(#cycles)

30 cycles => up to 230 ≈ 109 fold amplification Fig 5-20

Basics of PCR (polymerase chain reaction)

Using PCR to detect repeat expansions

Primer 1

Primer 2

Primer 1

Primer 2

Amplification by PCR

Amplification by PCR

Smaller PCR products

Larger PCR products

Person 1

Person 2

Gel electrophoresis sorts DNA molecules by size

– Gel electrophoresis separates DNA molecules based on size

– DNA sample is placed at one end of a porous gel

– Current is applied and DNA molecules move from the negative electrode toward the positive electrode

– WHY?

Copyright © 2009 Pearson Education, Inc.


DNA ladders or standards

• Whenever we run an agarose gel, DNA standards of known size are run as a guide to determine the molecular weight or amount of the DNA fragments in the experimental samples. These standards are often referred to as “ladders”.

• The standard we will using consists of the genome from lambda bacteriophage digested with the enzyme Hindlll.

• The resulting 7 fragments are 564, 2027, 2322, 4361, 6557, 9416, 23130 bp in size.

– (A 125 bp fragment is present but usually not seen.)

1 2 3

23,130 bp

9,416 bp

6,557 bp

4,361bp

2,322 bp 2,027 bp

Lane 1: lambda HIND lll standard Lane 2: unknown X Lane 3: unknown Y

We will using an agarose gel to determine both the size of the DNA

fragments in an unknown sample…

To determine size, compare how far bands have migrated relative to the standards – how big are the DNA molecules in sample X and Y?

We know this band contains 240 ng of DNA (assuming you loaded 10 ml of Hindlll lambda standard)

How much DNA would you estimate is in this lane in comparison to standard band?

Take that number and divide by amount of DNA you loaded onto gel = concentration of DNA in your sample.

Note: you divide by the total amount of DNA you loaded, not including the sample buffer or any water.

…and to get an idea of the amount of DNA in our samples by

comparing the intensity of the bands to those in the standards

DNA fingerprinting

PCR on multiple variable repeat

regions (several different

primer pairs) +

Gel electrophoresis

Fig 6-7

Who is the father?

A)F1

B)F2

DNA fingerprinting

PCR on multiple variable repeat

regions (several different

primer pairs) +

Gel electrophoresis

Fig 6-7

Who is guilty?

A)1 B)2 C)3

Table 16.1 Genomes 3 (© Garland Science 2007)

Diseases associated with repeat expansions

PCR can be used as diagnostic test for disease

• Huntington’s Disease: neurodegenerative disease caused by a polyglutamine expansion in the HTT protein (VIDEO)

Below is shown a region of genomic DNA containing repeat sequences.

You wish to generate the PCR product shown below.

Genomic DNA:

5’...CTGCGCGGCAGGTTGTAGTCAGCAGCAGCAGTGCGTATTTGAGTAGA...3’

3’...GACGCGCCGTCCAACATCAGTCGTCGTCGTCACGCATAAACTCATCT...5’

A: 5’-TCCAACATCA-3’ and 5’-AAATACGCA-3’

B: 5’-AGGTTGTAGT-3’ and 5’-ACGCATAAA-3’

C: 5’-ACTACAACCT-3’ and 5’-TGCGTATTT-3’

D: 5’-AGGTTGTAGT-3’ and 5’-AAATACGCA-3’

E: 5’-TCCAACATCA-3’ and 5’-TGCGTATTT-3’

Desired PCR Product:

5’-AGGTTGTAGTCAGCAGCAGCAGTGCGTATTT-3’

3’-TCCAACATCAGTCGTCGTCGTCACGCATAAA-5’

Which primers would you use?

Repeat

More than 3 million mobile elements in our genome!

(~45%)

Ge

ne

s

Mobile DNA elements = Transposable elements

DNA can move within and between chromosomes ~1940s – very controversial idea!

VIDEO

Barbara McClintock MacArthur “Genius” Award, 1981

Lasker Award, 1981

Nobel Prize, 1983

Summer of 1944: Cold Spring Harbor Labs, Long Island

Developed the transposon theory by studying corn kernel coloration.

Wild-Type

Mutant Revertants

Two general types of transposition

Fig 6-8

Cut&Paste Copy&Paste

DNA transposons Retrotransposons

Bacterial DNA transposons –

IS (insertion sequences)

Transposase

Fig. 10-9

Fig 6-9

Encodes

Catalyses DNA transposition

Transposition of a bacterial IS transposon

Fig 6-10

Encoded by Transposon

Cellular DNA repair enzymes

Cut target DNA

Cut Transposon

Creates target site direct repeats

You add a drug that inhibits cellular

DNA Ligase to a cell undergoing IS

element DNA transposition.

According to the model for DNA

transposition, which Target DNA

intermediate of the transposition

reaction should accumulate?

5’ 5’ 3’ 3’

3’ 3’ 5’ 5’

5’ 3’

3’ 5’

5’

3’

3’

5’

A:

B:

C:

D:

E:

DNA Transposition reaction

DNA transposons can increase in copy number

during DNA replication

Eukaryotic retrotransposons

Reverse Transcriptase

Fig 6-12

LTR: Long terminal repeat

NOTE: Similar to Retroviruses – but lack envelope proteins

Integrase

and

Encodes

Catalyze Retro-transposition

General mechanism of Retrotransposition

Based on Fig 6-12

Retrotransposon RNA

Retrotransposon DNA

Target DNA

1) Reverse Transcriptase makes DNA

copy

Encoded by Transposon

2) Integrase – inserts Retrotransposon

into target DNA

Fig 6-16

LINEs: - Long Interspersed Elements

- 21% of total human DNA (900,000!)

- ~6,000bp intact, but 99.99% are not

SINEs: - Short Interspersed Elements (Lack ORFs)

(a class of SINEs = Alu elements)

- rely on LINE protein for retrotransposition

- ~300bp

- ~13% of total human DNA (1,600,000!)

Common types of human retrotransposons

Alu Elements

• Over 1 million in genome

• 10.7% of genome

• Connected to many cancers, diabetes, hemophilia, etc.

• Can be used to trace human history

A: It is a DNA transposon.

B: It is a retrotransposon.

C: It could be either a DNA transposon

or a retrotransposon.

D: It is neither a DNA transposon or a

retrotransposon

You are studying a new mobile element in yeast, and want to determine if it is a

retrotransposon or a DNA transposon.

To do so, you perform an experiment: You treat one culture of growing yeast

with an RNA polymerase inhibitor while leaving a second culture untreated.

The next day, you find that the mobile element has transposed to new target

sites in the untreated culture, but did not in the treated culture.

What can you conclude from these data about the

mobile element?

RNA pol inhibitor

added untreated

transposition occurred

transposition blocked

Transposons are kept (mostly) silent

Strong silencing maintained at both the transcriptional and post

transcriptional levels: chromatin structure and RNAi (more on

this in future weeks)

When would it be

particularly important to

CONTROL transpositions?

1. Generation of gene families – duplications through homologous sites

for unequal crossing over

2. Creation of new genes - exon shuffling

3. Formation of complex regulatory regions that control gene expression

Mobile DNA elements and genome evolution

25% of our genome is “unclassified”!

Spacer DNA – really “junk”??

Role in gene expression? • complex transcriptional control regions affecting promoter activity

Role in cellular organization? • may impact structure of chromosomes and how DNA is organized

in the nucleus

Unknown genes - new proteins or non-coding RNAs? • Deep sequencing of RNA transcripts = lots of transcription

occurring in spacer DNA!

Provide space between genes to isolate the effect of mutation.

How can we study the function of a gene?

Gene cloning Allows study of the gene in separation from the remainder of the genome. Gene inactivation Allows study of the cellular/organismal effect of loss of gene function.

How do you isolate and propagate a piece of DNA (for example a gene)?

Genomic or Viral DNA

Vector (= plasmid or viral DNA that can replicate in a desired

organism - often E. coli)

Cut out piece of DNA

Insert into vector Amplify in

cells

(e.g. E. coli)

GAATTC

CTTAAG

5’

5’

3’

3’

G 3’ 5’ AATTC

CTTAA 5’ 3’ G

5’

5’

3’

3’

GGTACC

CCATGG

5’

5’

3’

3’

GGTAC 3’ 5’ C

C 5’ 3’ CATGG

5’

5’

3’

3’

Restriction enzymes cleave DNA at specific (usually palindromic) sequences

EcoRI

KpnI

Similar to Fig. 5-11

Similar to Table 5-1

Based on Fig. 4-1

Many bacteria contain restriction-modification systems to

“restrict” invasion by foreign DNA

GAATTC

CTTAAG

CH3

I

I

CH3

Bacterial genome DNA Invading DNA (e.g. virus)

GAATTC

CTTAAG

5’

5’

3’

3’

G 3’ 5’ AATTC

CTTAA 5’ 3’ G

5’

5’

3’

3’

5’

3’ 5’

3’

Modifying enzyme (methylase)

EcoRI

EcoRI

Where do restriction enzymes come from?

Fig. 5-12

Ligating a DNA fragment to a vector

Vector

DNA fragment

Obtaining bacterial clones with your recombinant plasmid

Fig. 5-14

Bacterial colonies containing your plasmid (only plasmid-containing bacteria will survive on

ampicillin)

Pick a colony - you have your

clone!

Plate on selective media (e.g. ampicillin)

What - in addition to a selectable marker gene - is required in

a plasmid to ensure that bacteria retain it through multiple

generations?

A: A telomere

B: An origin of replication

C: A restriction site

D: A gene for a modifying enzyme

How do you obtain a specific insert DNA (for example a gene)?

From a DNA source (e.g. genomic DNA)

- PCR

- Genomic library

From an RNA source (e.g. total cellular RNA)

- RT-PCR to create a cDNA library

Genomic DNA

Creating a genomic DNA insert using PCR

PCR

Ligation into vector

- Transform to bacteria,

- Select

see Fig 5-24

Primer 1

Primer 2

PCR product (e.g. a gene)

Creating a copy DNA (cDNA) clone from mRNA (RT-PCR)

mRNA

Primer 2

Reverse Transcriptase + dNTPs

mRNA

cDNA

5’ 3’ 5’

5’ 3’ 5’

3’

PCR

Primer 1

Primer 2

RNase H

cDNA 3’ 5’

3’ 5’ ds-cDNA 5’ 3’

- Ligate into vector


- Select

Similar to Fig 5-15

Restriction sites can be added via primer sequences

PCR

Primer 1

Primer 2

3’ 5’ PCR product 5’ 3’

For example:

EcoRI

BamHI

BamHI EcoRI

DNA

Restriction digest, Ligation into vector

BamHI EcoRI

It is sometimes critical to have a specific orientation of the insert in a vector, for

example when you want to express an inserted gene from a pre-existing promoter

in the plasmid.

What would be the orientation of the insert in the following cloning?

HindIII EcoRI

HindIII EcoRI

Insert Plasmid

HindIII: A AGCTT EcoRI: G AATTC

HindIII EcoRI HindIII EcoRI

A B

C: Mix of ‘A’ and ‘B’

D: Neither ‘A’ nor ‘B’

It is sometimes critical to have a specific orientation of the insert in a vector, for

example when you want to express an inserted gene from a pre-existing promoter

in the plasmid.

What would be the orientation of the insert in the following cloning?

HindIII EcoRI

HindIII EcoRI

Insert Plasmid

HindIII: A AGCTT EcoRI: G AATTC

HindIII EcoRI HindIII EcoRI

A B C: Mix of ‘A’ and ‘B’

D: Neither ‘A’ nor ‘B’

How do you know your bacterial clone contains your plasmid (and not e.g. self-ligated plasmid)?

Pick a colony (and expand in

selective medium)

Isolate plasmid

Restriction digest (e.g. BamHI)

Analyze by agarose gel

electrophoresis

Large DNA

Small DNA

Fig. 5.1

-

+

Stain with DNA-specific dye (e.g. EtBr)

Analysis by restriction digestion

Pick 5 clones (A-E); Restriction digest

isolated plasmids with BamHI;

Separate in agarose gel

4,000 bp

500 bp

4,000 bp

3,000 bp

2,000 bp

1,000 bp

A B C D E

Which clone (A-E) has the correct plasmid?

WHY DO ALL CELLS IN A SINGLE COLONY HAVE THE SAME PLASMID?

Pick 5 clones (A-E); Restriction digest

isolated plasmids with BamHI;

Separate in agarose gel

4,000 bp

500 bp

4,000 bp

3,000 bp

2,000 bp

1,000 bp

A B C D E

Which clone (A-E) has the

correct plasmid?

WHY DO ALL CELLS IN A SINGLE COLONY HAVE THE SAME PLASMID?

6-3

Creating a genomic library

Genomic DNA

Chop up with restriction enzyme

Transform to bacteria, select

- Each clone will have a plasmid with a specific genomic insert.

- Different clones contain different inserts.

Similar to Fig 5-17

Ligate with plasmid

Multiple plasmids, each with different DNA inserts!

DNA fragments

- Ligate to plasmid,


- Select (= cDNA library) see Fig 5-15

Creating a eukaryotic cDNA library

mRNAs

oligo-dT Primer

Reverse Transcriptase + dNTPs

mRNA

cDNA

5’ 3’

5’

5’ 3’ 5’

3’

AAAAAAA

AAAAAAA TTTTTTTT

Will anneal to all poly(A) tails!

RNase H (degrades RNA in RNA:DNA hybrid)

RNA oligo + RNA ligase

cDNA 5’ 3’ TTTTTTTT

ds-cDNA 5’ 3’ TTTTTTTT

Primer AAAAAAA

DNA polymerase + primer (annealing to 5’ end)

For example:

Remove cap 5’ 3’ AAAAAAA

Added to all mRNAs!

Finding the colony that contains a plasmid with your gene of interest in a library

32P-labeled probe (DNA or RNA) hybridizing to your gene of interest

Fig 5-16

Colony hybridization

What can you do with your gene clone?

Vector

Gene of interest

- Express protein (for example in bacteria) For studies of protein function/structure. For medical or nutritional production of protein. - Express gene, or mutant gene, in organism of origin For studies of gene expression and/or function. Gene therapy???

How can we study the function of a gene?

Gene cloning Allows study of the gene in separation from the remainder of the genome. Gene inactivation Allows study of the cellular/organismal effect of loss of gene function.

Gene inactivation in yeast

Gives resistance to G-418

Fig 5-39

Same principle used in

bacterial gene inactivation

1) Generate a DNA that contains regions

flanking the gene to be knocked out

2) Recombine the DNA into the genome

Genomic sequence

Conditional gene inactivation in mice using Cre/lox

Fig 5-42

Multiple steps of gene expression

Fig. 1-11

TRANSCRIPTION: Production of a

primary RNA transcript from a gene

The process of transcription

DNA Template strand

5’-to-3’ RNA strand growth

Incoming NTP

3’ 5’

5’ 3’

Based on Fig. 4-10

Base pairing

Release of pyrophosphate (PPi)

RNA Polymerase

Which of these nucleotides is used in Transcription?

(A) (B) (C)

(D)

RNA polymerase

Genomic DNA

?????

How does RNA polymerase know where to start transcription?

RNA polymerase

E. coli promoter

RNA polymerases recognize promoter sequences Prokaryote model –

5’ 3’

5’ 3’ DNA

+1

Sigma (s) factor

E. coli promoter

s factor binds conserved promoter elements (In normal E. coli genes: -10, -35)

RNA polymerase (a2bb’s)

E. coli RNA polymerase is a multi-subunit protein complex

+1

a2

b

b

Common eukaryotic RNA Polymerase II promoter elements

Transcription start (+1)

Upstream element: CpG islands upstream of start site

BRE: TFIIB Recognition Element

TATA: TATA box

Inr: Initiator element

DPE: Downstream Promoter Element

Note: Few (if any) Pol-II promoters contain all of these elements! None of these elements found in all Pol-II promoters!

Jim Kadonaga, UCSD

The three steps of transcription

5’

5’

3’

Template strand

Non-template strand RNA polymerase

DNA

RNA

Primary transcript Similar to

Fig. 4-11

Melts the DNA

RNA Pol synthesizes RNA 5’-to-3’

Termination releases RNA Pol and RNA

Bacterial RNA Polymerase

Eukaryotic mRNA

The three steps of transcription

5’

5’

3’

Template strand

Non-template strand RNA polymerase

DNA

RNA

Primary transcript Similar to

Fig. 4-11

Melts the DNA

RNA Pol synthesizes RNA 5’-to-3’

Termination releases RNA Pol and RNA

Fig. 7-8

Biochemical fractionation of eukaryotic nuclei identified three eukaryotic RNA polymerases

Which polymerase transcribes protein-coding genes?

a-amanitin inhibits transcription of protein-coding genes and RNA Polymerase II

Eukaryotic RNA Polymerases specialize in transcribing specific types of genes

Eukaryotic vs. Prokaryotic RNA Polymerases

RNA Polymerases contain multiple subunits

Fig. 7-10

Pol II large subunit C-

terminal domain (CTD):

- Multiple copies (52 in

human) of seven-amino acid

repeat, incl 3 serines.

- CTD phosphorylation is

critical for txn initiation.

RNA polymerase

A. RNA polymerase I

B. RNA polymerase II

C. RNA polymerase III

D. The majority of miRNAs are produced by RNA pol II, a minority by RNA pol III

E. RNA polymerases I, II and III are all used to produce miRNAs

The human genome encodes hundreds of 21-23 nucleotide RNAs that regulate

mRNA translation called miRNAs. You wish to identify the RNA polymerase(s)

responsible for transcription of miRNAs and pulse-32P-label all RNA in cells in the

presence of various concentrations of a-amanitin, isolate miRNAs and subject

them to gel electrophoresis. Based on the result, which is the polymerase used?

miRNAs

A. RNA polymerase I

B. RNA polymerase II

C. RNA polymerase III

D. The majority of miRNAs are produced by RNA pol II, a minority by RNA pol III

E. RNA polymerases I, II and III are all used to produce miRNAs

The human genome encodes hundreds of 21-23 nucleotide RNAs that regulate

mRNA translation called miRNAs. You wish to identify the RNA polymerase(s)

responsible for transcription of miRNAs and pulse-32P-label all RNA in cells in the

presence of various concentrations of a-amanitin, isolate miRNAs and subject

them to gel electrophoresis. Based on the result, which is the polymerase used?

miRNAs

7-3

How do we know the DNA elements that control transcription?

Based on Fig. 7-13

Assay for reporter gene expression

(in vitro or in vivo)

Reporter Assay:

1. Northern blotting (Detects RNA levels using probes).

2. Easily assayed gene products.

For example:

a. X-gal galactose + BLUE

b-galactosidase (LacZ)

b. luciferin light emission

luciferase

c. GFP - protein that fluoresces green under UV light

How can we detect reporter gene expression?

How do you detect a specific cellular RNA?

+

-

1. isolate total RNA

4. Detect using radiolabeled

antisense probe

3. transfer to membrane,

fix, hybridize

2. denature and separate RNA

by electrophoresis

Experimental detection of RNA by Northern blotting

FOR EXAMPLE: Northern blot for b-globin mRNA in differentiating erythrocytes

Hours after differentiation:

Fig. 5-27

1. Northern blotting (Detects RNA levels using probes).

2. Easily assayed gene products.

For example:

a. X-gal galactose + BLUE

b-galactosidase (LacZ)

b. luciferin light emission

luciferase

c. GFP - protein that fluoresces green under UV light

How can we detect reporter gene expression?

Osamu Shimomura

Roger Tsien, UCSD

Nobel Prize

Chemistry 2008

Which of the following 32P-labeled DNA probes could be used

to detect the mRNA shown above?

5’CGGUAAAUGGUUAGUCGAUGGGUUCUCGAUGAGC3’ mRNA:

A: 5’-32P-CCCAAGAGCTACT-3’

B: 5’-32P-GGGTTCTCGATGA-3’

C: 5’-32P-AGTAGCTCTTGGG-3’

D: 5’-32P-TCATCGAGAACCC-3’

Which specific promoter

sequences are required for

transcription? Linker scanning

mutations

Reporter mRNA

+1 -100

Unrelated mRNA

WT

Reporter mRNA

+1 -100

Unrelated mRNA

WT

D

lecture 3 - genomes and transcription

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