Lecture 5 Flashcards
most cancers are recessive or dominant??
Recessive
Are oncogenes recessive or dominant?
Dominant
Viral oncogenes exert what a effect ?
Dominant - Transformation
Rao and Johnson
- 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
What are the arguments for yes or no tumour suppressor genes exist ?
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
Experiment to provide evidence for dominant or recessive alleles in cancer cells ?
Combine normal cell and cancer cell.
> If Alleles are dominant hybrid will be tumorigenic
> If hybrid cells non -tumorigenic cancer alleles are recessive.
Retinoblastoma
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
What cancer type gives an insight into tumour suppression ?
Retinoblastoma
Knudson’s 1-hit/2 hit Hypothesis
also known as the two-hit hypothesis is the hypothesis that most genes require two mutations to cause a phenotypic change.
Evidence for Knudson’s 1-hit/2 hit Hypothesis
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
How do you explain the frequency of loss of second wt allele?
- 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
Cytogenetics –
-banding analysis of chromosomes missing critical region
Stephen Friend et al
- Cloning of the Rb gene confirms existence of genes with properties of tumour suppressors
loss of heterozygosity generates what type of cells ??
Cells that lack a critical tumour suppressor gene
Cytogenetic analysis facilitated the identification of what gene
Rb- Retinoblastoma
In RB which chromosome is missing a band?
13 - critical region
Zymography proved what ?
Loss of heterozygosity in 13q14 (14 is the region, 13 the chromosome) in Rb
Sparkes et al
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
Stephen Friend et al theory
Cloning of the Rb gene confirms existence of genes with properties of tumour suppressors
Stephen friend et al experiment
Northern blot -RNA from patients fragment it and probe radio-labelled that binds to target sequence
-gene missing - DNA fragment missing in Rb
How do you find tumour suppressor genes ? methods?
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
How would you profile and establish allelic deletions required for each chromosome ?
- 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
