Genetics of Cancer. Fig. 11-12 Signaling cell DNA Nucleus Transcription factor (activated) Signaling molecule Plasma membrane Receptor protein Relay proteins

Genetics of Cancer

Fig. 11-12Signaling cell

DNA

Nucleus

Transcriptionfactor(activated)

Signaling molecule Plasma

membraneReceptorprotein

Relayproteins

TranscriptionmRNA

Newprotein

Translation

Target cell

2

1

3

4

5

6

Signal Transduction:

Way in which a cell can respond to signals from its environment

Results in a change in which genes are expressed (turned on)

Fig. 11-20b

Growth-inhibiting factor

Protein thatinhibitscell division

Translation

Normal productof p53 gene

Receptor

Relayproteins


Nonfunctional transcriptionfactor (product of faulty p53tumor-suppressor gene) cannot trigger transcription

Transcription

Protein absent(cell divisionnot inhibited)

Normal tumor-suppressor genes prohibit cell division

Fig. 11-20aGrowth factor

Protein thatStimulatescell division

Translation

Nucleus

DNA

Target cell

Normal productof ras gene

Receptor

Relayproteins


Hyperactiverelay protein(product ofras oncogene)issues signalson its own

Transcription

Ras is an oncogene (cancer gene) the normal form of the gene is a proto-oncogene

Oncogenes STIMULATE cell division

Fig. 11-18b

Mutated tumor-suppressor geneTumor-suppressor gene

Defective,nonfunctioningprotein

Normalgrowth-inhibitingprotein

Cell divisionunder control

Cell division notunder control

6

Progression of Colon Cancer

Both alleles of BRCA1 or both alleles of BRCA2 must be mutant for cancer to develop.

Why would in follow a dominant inheritance pattern?

A tissue comprised of billions of cells heterozygous for BRCA1 or BRCA2

8

Your (my) probability of winning the lottery is very small. The probability that someone will win it is very large.

One of the key tools in DNA technology is the restriction enzyme

Where do these restriction enzymes come from????

What is their natural function???

How can we use them???

Recombinant DNA

• DNA from 2 sources combined– Can be used to clone genes– Used to produce a particular protein

E. coli bacterium

Plasmid

Bacterialchromosome

Gene of interest

DNA

Cell with DNAcontaining geneof interest

Isolateplasmid

IsolateDNA

1 2

A plasmid is a small circular piece of DNA found in some bacterial cells

Separate from main chromosome

May have genes that give the bacteria an advantage in certain circumstances

Bacteria can take up plasmids from their environment

E. coli bacteriumPlasmid

Bacterialchromosome

Gene of interest

DNA


Gene of interest

Isolateplasmid

IsolateDNA

Cut plasmidwith enzyme Cut cell’s DNA

with same enzyme

1

2

34


Bacterialchromosome

Gene of interestDNA


Gene of interest

Isolateplasmid

IsolateDNA

Cut plasmidwith enzyme

Cut cell’s DNAwith same enzyme

1

2

3

4

Combine targeted fragmentand plasmid DNA

5


Bacterialchromosome

Gene of interestDNA


Gene of interest

Isolateplasmid

IsolateDNA

Cut plasmidwith enzyme

Cut cell’s DNAwith same enzyme

1

2

3

4

RecombinantDNAplasmid

Geneof interest

Combine targeted fragmentand plasmid DNA

Add DNA ligase,which closesthe circle withcovalent bonds

5

6


Geneof interest

Recombinantbacterium

Put plasmidinto bacterium

7

RecombinantDNAplasmid Gene

of interest


Cloneof cells-gene of interest has also been cloned

Put plasmidinto bacteriumby transformation

Allow bacteriumto reproduce

7

8

7


Geneof interest


Cloneof cells

Genes or proteinsare isolated from thecloned bacterium

Harvestedproteinsmay be used directly

Examples ofprotein use

Put plasmidinto bacteriumby transformation

Allow bacteriumto reproduce

8

7

Genes may be insertedinto other organisms

Examples ofgene use

9

Important to use the same restriction enzyme to cut each source of DNA

This allows complementary sticky ends to be created that can later base-pair to combine the DNA

Restriction enzymerecognition sequence

1

2

DNA

Restriction enzymecuts the DNA intofragments

Sticky end


1

2

DNA


Sticky end

3

Addition of a DNAfragment fromanother source


1

2

DNA


Sticky end

3


4

Two (or more)fragments sticktogether bybase-pairing


1

2

DNA


Sticky end

3


4

Two (or more)fragments sticktogether bybase-pairing

DNA ligasepastes the strands

RecombinantDNA molecule5

1. Plasmid DNA is isolated

2. DNA containing the gene of interest is isolated

3. Plasmid DNA is treated with restriction enzyme that cuts in one place, opening the circle

4. DNA with the target gene is treated with the same enzyme and many fragments are produced

5. Plasmid and target DNA are mixed and associate with each other

Copyright © 2009 Pearson Education, Inc.

Steps in cloning a gene

6. Recombinant DNA molecules are produced when DNA ligase joins plasmid and target segments together

7. The recombinant DNA is taken up by a bacterial cell

8. The bacterial cell reproduces to form a clone of cells


Steps in cloning a gene

Problem: if we’re trying to get a bacterium (prokaryote) to make our proteins, bacteria do not have introns… so, they can’t remove them

Solution: Use reverse transcriptase (found in retroviruses) to make DNA from mature mRNA

Use restriction enzymes to break DNA into manageable sized pieces

that we can separate

What can we tell from this?

• It can be used to compare the DNA from different organisms

• Used to detect disease alleles• Used to “match” DNA samples

– Determine parentage– Crime scene forensics

Fig. 12-11

Crime sceneDNA isolated1

Suspect 1 Suspect 2

DNA of selectedmarkers amplified

2

Amplified DNA compared

3

PCR is used to amplify DNA sequences

• http://learn.genetics.utah.edu/content/labs/pcr/

– Mix ingredients in a thermocycler• What do you need to make lots of copies of DNA?


Detecting disease alleles

Fig. 12-14a

STR site 1

Crime scene DNA

STR site 2

Suspect’s DNA

Number of short tandemrepeats match

Number of short tandemrepeats do not match

Fig. 12-14b

Crime sceneDNA

Suspect’sDNA

Cycle 1yields 2 molecules

21 3

GenomicDNA



3 5 3 5 3 5

Targetsequence

Heat toseparateDNA strands

Cool to allowprimers to formhydrogen bondswith ends oftarget sequences

35

3 5

35

35 35

Primer New DNA

5

DNApolymerase addsnucleotidesto the 3 endof each primer

5


GenomicDNA

3 5 3 5 3 5

Targetsequence

Heat toseparateDNA strands

Cool to allowprimers to formhydrogen bondswith ends oftarget sequences

35

3 5

35

35 35

Primer New DNA

5

DNApolymerase addsnucleotidesto the 3 endof each primer

215

3



Documents

Genetics of Cancer. Fig. 11-12 Signaling cell DNA Nucleus Transcription factor (activated) Signaling molecule Plasma membrane Receptor protein Relay proteins