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35 Cancer- part 2Lecture Outline, 11/30/05
• Finish Cancer genetics– Review Oncogenes and proto-oncogenes– Tumor Suppressor genes
• Normally inhibit cell growth.• Allow cell growth when damaged or deleted.
– Mutator genes– The multi-step model of cancer
• Cloning a cancer gene: BRCA1
Case Study: BRCA1
Narod, Steven A. BRCA1 and BRCA2: 1994 andBeyond. Nature Reviews (2004), 670.
Probably involved inDNA repair pathways
Would this be a tumorsuppressor or anoncogene?
BRCA1: DNA Repair
Kennedy, Richard D. The Role of BRCA1 in the CellularResponse to Chemotherapy. Journal of National CancerInstitute (2004), 1660.
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Finding the Cancer GeneBRCA1
• 1980’s: found several families that werepredisposed to breast cancer
• Studied 23 breast cancer families– Early onset– Frequent bilateral disease– Male relatives with breast cancer
• 1990: linked the disease to a marker onChromosome 17q21– D17S74 - 183rd marker used!– Initial candidate region spanned half the
chromosome (hundreds of possible genes . . .)
124
8
2 , 8 4 , 8 1 , 2
1 , 8 2 , 4
Linkage study
Loci far apart
AB
ab
AB
Ab
aB
ab
Recombinants: Ab and aB
Loci close together
AB ab
AB ab AB ab
No recombinants between A and B
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• Even when a disease gene has not yet beencloned an abnormal allele can be diagnosedwith reasonable accuracy if a closely linkedRFLP marker has been found
Figure 20.15
RFLP markerDNA
Restrictionsites
Disease-causingallele
Normal allele
Restriction enzymes cut DNA at particular sequences
• Two alleles of a gene may producerestriction fragments with differentlengths.
Figure 20.9
Normal β -globin allele
Sickle-cell mutant β-globin allele
175 bp 201 bp Large fragment
DdeI DdeI DdeI DdeI
DdeI DdeI DdeI
376 bp Large fragment
DdeI restriction sites intwo alleles of theβ-globin gene.
Electrophoresisshows that thefragments havedifferent lengths
Normalallele
Sickle-cellallele
Largefragment
201 bp175 bp
376 bp
Dde1 cuts at thesequence
C|TNAG
GANT|C
4
DNA + restrictionenzyme Restriction
fragments I II III
I Normalβ-globinallele
II Sickle-cellallele
III Heterozygote
Preparation ofrestrictionfragments
Gelelectrophoresis
Blotting: transfer to anylon membrane
Gel
Sponge
Alkalinesolution
Nitrocellulosepaper (blot)
Heavyweight
Papertowels
1 2 3
Figure 20.10
Radioactivelylabeled probefor is addedto solution ina plastic bag
Probe hydrogen-bonds to fragmentscontaining thecomplementary DNAsequence
Fragment fromsickle-cellβ-globin allele
Fragment fromnormal β-globinallele
Paper blot
Film overpaper blot
Hybridization withradioactive probe.
Autoradiography.
I II IIII II III
4 5
How would youmake the probe?
Linkage study
Disease Allele “A”*
DNA probe
Normal Allele “B”DNA probe
AA AB BB
What next?
Identifyrecombinants
Try moremarkers
Test morefamilies
5
Recombination
246
453
121
864
243
864
Marker 1Marker 2Marker 3
Occasionally there isa crossover duringmeiosis
To find those rarecrossovers, theyneeded manyfamilies with inheritedbreast cancer
This individualshows that it is notnear Marker3
Mapping BRCA1
• Larger study• 214 breast cancer families
– Region narrowed to 8 cM• That is still a 600,000 nucleotide region
• Step 2: Positional cloning
Figure 20.3
Restriction site
DNA 5′3′ 5′
3′G A A T T CC T T A A G
Sticky endFragment from differentDNA molecule cut by thesame restriction enzyme
One possible combination
Recombinant DNA molecule
GC T T A A
A A T T CG
A A T T C
C T T A AG G
G GA A T T C A A T T CC T T A A G C T T A A G
Using a restriction enzyme and DNAligase to make recombinant DNA
Cut DNA withRestrictionenzyme, leavingoverhanging ends
1
Base pairing of sticky ends produces various combinations.
2
DNA ligaseseals the strands.
3
6
Transform therecombinantplasmid into E.coli
To produce a “library” of different DNA fragments
Order and Sequence theclones
Contig construction
1 Probe a large insertlibrary to identify aclone containing themarker linked to thetrait. sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
7
2 Probe a large insertlibrary to identifyclones containing thesequence of the endsof the first clone
Contig construction
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
3 These clones must overlap thefirst clone. ie they have some ofthe same DNA - and hopefully alsosome not in the first clone
Contig construction
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
4 Again, probe the large insert libraryto identify clones containing thesequence of the ends of these clones.
Contig construction
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
4 Again, these clones must overlap theexisting clones. ie they have some of thesame DNA - and hopefully also somenew sequence
Contig construction
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
8
In this way we build up a CONTIG - aseries of overlapping clones centred onour region of interest.
Contig construction
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
Results of sequencing
– Found 65 expressed genes– Looked for sequence differences between family
members with and without cancer
BRCA1 found in 1994Science. 1994 Oct 7;266(5182):66-71.
A strong candidate for the breast and ovarian cancersusceptibility gene BRCA1.Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K,Tavtigian S, Liu Q, Cochran C, Bennett LM, Ding W, et al.Department of Medical Informatics, University of Utah MedicalCenter, Salt Lake City 84132.A strong candidate for the 17q-linked BRCA1 gene, which influencessusceptibility to breast and ovarian cancer, has been identified bypositional cloning methods. Probable predisposing mutations havebeen detected in five of eight kindreds presumed to segregate BRCA1susceptibility alleles.
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How would youmake the probe?
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