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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

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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

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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

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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

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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

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Base pairing of sticky ends produces various combinations.

2

DNA ligaseseals the strands.

3

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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/

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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/

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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?