Lecture 7

Question 1

P53

Answer 1

• P53 is mutated in a large proportion of cancers
• Tumour suppressor gene
• Short lived
• Induces apoptosis after prolonged expression
• P53 is a short-lived protein whose steady state level is controlled by regulated proteolysis

Question 2

Discovery of P53 - Lane and Harlow

Answer 2

• What is the molecular basis for the decision to undertake DNA replication ??
• Use polyomavirus SV40
• Viral infection promotes DNA replication in host
• Theory – if mechanism understood might tell us hoe mammalian cells control DNA replication

Question 3

Lane and Harlow Experiment chosen stain

Answer 3

Peroxidase Staining

Question 4

Cells transformed with SV40

Answer 4

are very immunogenic and reside in the nucleus

Question 5

Viral protein required for transformation and inducing DNA replication

Answer 5

Large T

Question 6

Lane and Harlow Experiment

Answer 6

• Every protein has methionine – starting a.a – incubate -lables all proteins
• Made antibodies to Large T
• Labelled fibroblasts with 35S methionine
• Immunoprecipitated Large T
• Autoradiogram
• Protein in one lane on its own > SV40 large T < cellular protein that specifically associates with large T

Question 7

Moshe Oren

Answer 7

• Clone P53 in tissue culture cells
• Looking at anchorage independent growth
• Increase in Foci in the middle one suggest oncogene ?!
• But 3rd – clearly a tumour suppressor gene

Question 8

P53 in cancer

Answer 8

• Huge incidence of P53 in cancer cells
• Mutated in pretty much every tissue- most Common mutated gene
• Not your typical TS gene > mouse model TS deletion > embryonic lethal
• Loss of heterozygosity

Question 9

Knudesn conundrum

Answer 9

notion of one hit or two hits