Lecture 5

Question 1

most cancers are recessive or dominant??

Answer 1

Recessive

Question 2

Are oncogenes recessive or dominant?

Answer 2

Dominant

Question 3

Viral oncogenes exert what a effect ?

Answer 3

Dominant - Transformation

Question 4

Rao and Johnson

Answer 4

• Sendai virus
• All chromosomes in one nucleus – chromosomes don’t know where they came from
• Fuse Monkey kidney cells with NIH3T3
• Cancer cell phenotype is recessive

Question 5

What are the arguments for yes or no tumour suppressor genes exist ?

Answer 5

YEs- - loss of growth suppressor gene more likely than gain of function ras mutations
NO- loss of both alleles of putative growth suppressor
genes unlikely
- had to lose both copies

Question 6

Experiment to provide evidence for dominant or recessive alleles in cancer cells ?

Answer 6

Combine normal cell and cancer cell.
> If Alleles are dominant hybrid will be tumorigenic
> If hybrid cells non -tumorigenic cancer alleles are recessive.

Question 7

Retinoblastoma

Answer 7

Current treatment is to remove the organ

• sporadic or genetically inherited
• familial > bilateral loss of both eyes
• Recessive trait
• Implies single gene disease with a recessive allele
• Increase in non retinal tumours in patients with retinoblastoma
• Cancer predisposing disease

Question 8

What cancer type gives an insight into tumour suppression ?

Answer 8

Retinoblastoma

Question 9

Knudson’s 1-hit/2 hit Hypothesis

Answer 9

also known as the two-hit hypothesis is the hypothesis that most genes require two mutations to cause a phenotypic change.

Question 10

Evidence for Knudson’s 1-hit/2 hit Hypothesis

Answer 10

Take data of patients with unilateral or bilateral Rb over long period of time

• Semilog plot
• Percent of cases not yet diagnosed at the indicated age
• Plot data to know if a chemical reaction involves one reactant or two reactants
• Rate of reaction proportional to the conc of reactants
• Semi log plot – straight line if one reactant
• Curve if two reactants (Unilateral)
• Unilateral tumours- two independent events has to occur
• Only one hit to genome in bilateral – inherited the first hit – second hit happens they get cancer

Question 11

How do you explain the frequency of loss of second wt allele?

Answer 11

• Homologous recombination
• Allow sister chromosomes to recombine and swap arms essentially
• Alleles between cells are different
• Swapping over occurs then segregation
• Line up on mitotic spindle – multiple combinations
• Loss of heterozygosity in daughter cells- generates cells without tumour suppressor genes

Question 12

Cytogenetics –

Answer 12

-banding analysis of chromosomes missing critical region

Question 13

Stephen Friend et al

Answer 13

• Cloning of the Rb gene confirms existence of genes with properties of tumour suppressors

Question 14

loss of heterozygosity generates what type of cells ??

Answer 14

Cells that lack a critical tumour suppressor gene

Question 15

Cytogenetic analysis facilitated the identification of what gene

Answer 15

Rb- Retinoblastoma

Question 16

In RB which chromosome is missing a band?

Answer 16

13 - critical region

Question 17

Zymography proved what ?

Answer 17

Loss of heterozygosity in 13q14 (14 is the region, 13 the chromosome) in Rb

Question 18

Sparkes et al

Answer 18

D esterase – hydrolyses esters
But on chromosome 13 in region 14 (which is lost in Rb)
2 alleles of esterase gene and encode proteins of slightly different length
Zymography – form of gel electrophoresis proteins not denatured, impregnate gel with substrate of an enz and if they interact you can see in the gel
In Rb- notice that in tumour tissue always one isoform of D esterase people who are heterozygotes normal compared to tumour – loss of heterozygosity – proves homologous recombination

Question 19

Stephen Friend et al theory

Answer 19

Cloning of the Rb gene confirms existence of genes with properties of tumour suppressors

Question 20

Stephen friend et al experiment

Answer 20

Northern blot -RNA from patients fragment it and probe radio-labelled that binds to target sequence
-gene missing - DNA fragment missing in Rb

Question 21

How do you find tumour suppressor genes ? methods?

Answer 21

Loss of heterozygosity >

• RFLP- restriction fragment length polymorphism
• ECOR1 cleavable sites
• Sequence recognised by probe
• Southern blot analysis
• LOH at particular locus
• Cut with variety of restriction enz

Question 22

How would you profile and establish allelic deletions required for each chromosome ?

Answer 22

• Look for LOH
• RFLP analysis has largely been superseded by single nucleotide polymorphism analysis
• PCR based approach
• 3 million SNPS in human genome
  Easier to look at single nucleotide