35 Cancer- part 2. Lecture Outline, 11/30/05. BRCA1: DNA Repair. Case Study: BRCA1

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1 5 Cancer- part Lecture Outline, /0/05 Finish Cancer genetics Review Oncogenes and proto-oncogenes Tumor Suppressor genes Normally inhibit cell growth. llow cell growth when damaged or deleted. Mutator genes The multi-step model of cancer Cloning a cancer gene: RC Case Study: RC Probably involved in DN repair pathways RC: DN Repair Would this be a tumor suppressor or an oncogene? Narod, Steven. RC and RC: 99 and eyond. Nature Reviews (00), 670. Kennedy, Richard D. The Role of RC in the Cellular Response to Chemotherapy. Journal of National Cancer Institute (00), 660.

2 Finding the Cancer Gene RC 980 s: found several families that were predisposed to breast cancer Studied breast cancer families Early onset Frequent bilateral disease Male relatives with breast cancer 990: linked the disease to a marker on Chromosome 7q D7S7-8rd marker used! Initial candidate region spanned half the chromosome (hundreds of possible genes...) 8 Linkage study, 8,, 8, 8, Loci far apart Loci close together a b a b b a a b Recombinants: b and a a b a b No recombinants between and

3 Even when a disease gene has not yet been cloned an abnormal can be diagnosed with reasonable accuracy if a closely linked RFLP marker has been found Restriction enzymes cut DN at particular sequences DN RFLP marker Restriction sites Disease-causing Figure 0.5 Normal Two s of a gene may produce restriction fragments with different lengths. Normal β -globin 75 bp 0 bp Large fragment DdeI restriction sites in two s of theβglobin gene. DdeI DdeI DdeI DdeI Sickle-cell mutant β-globin 76 bp Large fragment DdeI DdeI DdeI Electrophoresis shows that the fragments have different lengths Large fragment Normal Sickle-cell 76 bp Dde cuts at the sequence C TNG GNT C 0 bp 75 bp Figure 0.9

4 I Normal β-globin DN + restriction enzyme II Sickle-cell III Heterozygote Restriction fragments I II III Sponge lkaline solution Preparation of Gel restriction electrophoresis fragments Figure 0.0 Nitrocellulose paper (blot) Gel Heavy weight Paper towels lotting: transfer to a nylon membrane Radioactively labeled probe for is added to solution in a plastic bag I II III Probe hydrogenbonds to fragments containing the complementary DN sequence Fragment from sickle-cell β-globin Fragment from normal β-globin Paper blot Hybridization with 5 radioactive probe. How would you make the probe? I II III utoradiogra phy. Film over paper blot Disease Normal Linkage study * DN probe DN probe llele llele What next? Test more families Try more markers Identify recombinants

5 Recombination Mapping RC Marker Marker Marker This individual shows that it is not near Marker Occasionally there is a crossover during meiosis 5 To find those rare crossovers, they needed many families with inherited breast cancer Larger study breast cancer families Region narrowed to 8 cm That is still a 600,000 nucleotide region Step : Positional cloning Using a restriction enzyme and DN ligase to make recombinant DN Restriction site Cut DN with Restriction enzyme, leaving overhanging ends ase pairing of sticky ends produces various combinations. DN 5 G T T C C T T G 5 G C T T T T C G Sticky end T T C G G C T T Fragment from different DN molecule cut by the same restriction enzyme DN ligase seals the strands. G T T C G T T C C T T G C T T G One possible combination Figure 0. Recombinant DN molecule 5

6 Transform the recombinant plasmid into E. coli Order and Sequence the clones To produce a library of different DN fragments Contig construction Probe a large insert library to identify a clone containing the marker linked to the sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/iol5/ trait. 6

7 Contig construction Contig construction Probe a large insert library to identify clones containing the sequence of the ends of the first clone sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/iol5/ These clones must overlap the first clone. ie they have some of the same DN - and hopefully also some not in the first clone sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/iol5/ Contig construction Contig construction gain, probe the large insert library to identify clones containing the sequence of the ends of these clones. sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/iol5/ gain, these clones must overlap the existing clones. ie they have some of the same DN - and hopefully also some new sequence sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/iol5/ 7

8 Contig construction Results of sequencing Found 65 expressed genes Looked for sequence differences between family members with and without cancer In this way we build up a CONTIG - a series of overlapping clones centred on our region of interest. sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/iol5/ RC found in 99 Science. 99 Oct 7;66(58):66-7. strong candidate for the breast and ovarian cancer susceptibility gene RC. Miki Y, Swensen J, Shattuck-Eidens D, Futreal P, Harshman K, Tavtigian S, Liu Q, Cochran C, ennett LM, Ding W, et al. Department of Medical Informatics, University of Utah Medical Center, Salt Lake City 8. strong candidate for the 7q-linked RC gene, which influences susceptibility to breast and ovarian cancer, has been identified by positional cloning methods. Probable predisposing mutations have been detected in five of eight kindreds presumed to segregate RC susceptibility s. 8

9 How would you make the probe? 9