genetics of cancer. fig. 11-12 signaling cell dna nucleus transcription factor (activated) signaling...
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Genetics of Cancer
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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)
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Fig. 11-20b
Growth-inhibiting factor
Protein thatinhibitscell division
Translation
Normal productof p53 gene
Receptor
Relayproteins
Transcriptionfactor(activated)
Nonfunctional transcriptionfactor (product of faulty p53tumor-suppressor gene) cannot trigger transcription
Transcription
Protein absent(cell divisionnot inhibited)
Normal tumor-suppressor genes prohibit cell division
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Fig. 11-20aGrowth factor
Protein thatStimulatescell division
Translation
Nucleus
DNA
Target cell
Normal productof ras gene
Receptor
Relayproteins
Transcriptionfactor(activated)
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
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Fig. 11-18b
Mutated tumor-suppressor geneTumor-suppressor gene
Defective,nonfunctioningprotein
Normalgrowth-inhibitingprotein
Cell divisionunder control
Cell division notunder control
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Progression of Colon Cancer
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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.
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One of the key tools in DNA technology is the restriction enzyme
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Where do these restriction enzymes come from????
What is their natural function???
How can we use them???
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Recombinant DNA
• DNA from 2 sources combined– Can be used to clone genes– Used to produce a particular protein
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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
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E. coli bacteriumPlasmid
Bacterialchromosome
Gene of interest
DNA
Cell with DNAcontaining geneof interest
Gene of interest
Isolateplasmid
IsolateDNA
Cut plasmidwith enzyme Cut cell’s DNA
with same enzyme
1
2
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E. coli bacteriumPlasmid
Bacterialchromosome
Gene of interestDNA
Cell with DNAcontaining geneof interest
Gene of interest
Isolateplasmid
IsolateDNA
Cut plasmidwith enzyme
Cut cell’s DNAwith same enzyme
1
2
3
4
Combine targeted fragmentand plasmid DNA
5
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E. coli bacteriumPlasmid
Bacterialchromosome
Gene of interestDNA
Cell with DNAcontaining geneof interest
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
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RecombinantDNAplasmid
Geneof interest
Recombinantbacterium
Put plasmidinto bacterium
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RecombinantDNAplasmid Gene
of interest
Recombinantbacterium
Cloneof cells-gene of interest has also been cloned
Put plasmidinto bacteriumby transformation
Allow bacteriumto reproduce
7
8
7
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RecombinantDNAplasmid
Geneof interest
Recombinantbacterium
Cloneof cells
Genes or proteinsare isolated from thecloned bacterium
Harvestedproteinsmay be used directly
Examples ofprotein use
Put plasmidinto bacteriumby transformation
Allow bacteriumto reproduce
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7
Genes may be insertedinto other organisms
Examples ofgene use
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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
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Restriction enzymerecognition sequence
1
2
DNA
Restriction enzymecuts the DNA intofragments
Sticky end
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Restriction enzymerecognition sequence
1
2
DNA
Restriction enzymecuts the DNA intofragments
Sticky end
3
Addition of a DNAfragment fromanother source
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Restriction enzymerecognition sequence
1
2
DNA
Restriction enzymecuts the DNA intofragments
Sticky end
3
Addition of a DNAfragment fromanother source
4
Two (or more)fragments sticktogether bybase-pairing
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Restriction enzymerecognition sequence
1
2
DNA
Restriction enzymecuts the DNA intofragments
Sticky end
3
Addition of a DNAfragment fromanother source
4
Two (or more)fragments sticktogether bybase-pairing
DNA ligasepastes the strands
RecombinantDNA molecule5
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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
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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
Copyright © 2009 Pearson Education, Inc.
Steps in cloning a gene
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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
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Use restriction enzymes to break DNA into manageable sized pieces
that we can separate
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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
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Fig. 12-11
Crime sceneDNA isolated1
Suspect 1 Suspect 2
DNA of selectedmarkers amplified
2
Amplified DNA compared
3
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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?
Copyright © 2009 Pearson Education, Inc.
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Detecting disease alleles
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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
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Fig. 12-14b
Crime sceneDNA
Suspect’sDNA
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Cycle 1yields 2 molecules
21 3
GenomicDNA
Cycle 3yields 8 molecules
Cycle 2yields 4 molecules
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
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Cycle 1yields 2 molecules
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
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Cycle 3yields 8 molecules
Cycle 2yields 4 molecules