Lecture 2 Cellular Oncogenes Flashcards Preview

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Flashcards in Lecture 2 Cellular Oncogenes Deck (38)
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1

Who predicted and discovered reverse transcriptase

Temin suggested the idea of an enzyme capable of converting RNA back to DNA before it could be transcribed and translated into protein. Along with Baltimore he then discovered this enzyme reverse transcriptase

2

The G12V transition in cRas is sufficient to create an oncogene T or F

T – this is sufficient to cause tumours in nude mice

3

What are the three effects of tumour viruses on cells in vitro

An increase thickness of the cell layer a loss of contact inhibition whereby cells no longer stop proliferating after they contact each other and finally the adoption of a rounded morphology

4

How does oncogenic cRas differ from the proto-oncogene

There is a single base pair change in the DNA that switches guanine to thymine. This results in an amino acid substitution that sees glycine 12 swapped to valine (G12V).

5

Subsequent work carried out by Michael Wigler set out to identify the sequence that caused transformation of cells. Describe the methodology Wigler used to characterise this oncogene

Wigler repeatedly carry out rounds of transformation of fibroblasts to see which fractions cause transformation. A mutagen was initially used to transform initial fibroblasts which formed foci in vitro on agar. The chromosome fragments from these foci were extracted and an essential bacterial gene was chemically attached to an end of all the DNA fragments from transformed foci cells. These newly ligated fragments containing the bacterial gene were aliquoted and CaPO4 transfected into wild type fibroblasts. The idea was that foci would only form in the newly transfected cells that received the aliquot(s) containing the oncogene. Then they repeatedly fragmented and extracted the DNA from the foci of the newly transfected fibroblasts and aliquoted it into several solutions. Bacteria that were lacking the essential gene that had been attached to the chromosomal fragments were then transformed with the DNA extracted from the transfected fibroblasts. Only bacteria receiving fractions containing the bacterial gene linked DNA would have been able to grow these would possess enriched levels of the oncogene. This was repeated several times to purify the oncogene.

6

What is the Ras protein and what are the effects of its mutation in terms of its function

Ras proteins are GTPases that bind guanine nucleotides. They need glycine 12 and glutamine 61 for phosphorylation and GTP binding. Glycine or glutamine transitions results in c-Ras adopting the active conformation in the absence of GTP resulting in constitutive activity and a subsequent overproliferation.

7

What do Bob Weinberg’s experiments mean in terms of the requirement of a virus for tumorigenesis

Weinberg’s experiments show that a mutagen alone is sufficient to cause transformation of cells independent of a virus

8

How can you alter the selectivity of a DNA probe in southern blotting

Changing the temperature will affect how easily the probe will bind to the target DNA higher temperatures will require the probe to be more specific. You can also change the salt solution too to vary the specificity of the probe

9

The myc transcription factors are another example of proto-oncogenes. What are the 3 types of myc

C-myc – gives rise to viral myc (v-myc) N-myc – largely expressed in the brain and finally L-myc

10

How are myc genes referred to when they are present in a viral genome

V-myc

11

What happens once RSV is inside the cell

Once inside the cell the RSV RNA genome is converted back to DNA by reverse transcriptase. This DNA can then incorporate into the genome of the host cell. Here is can be transcribed by the host cells machinery to RNA where it can then be translated into the proteins required to form a new viral particle.

12

Other than translocation what other change in regulation can cause the proto-oncogene to oncogene transition. How can this be observed experimentally

Amplification of genes can cause tumorigenicity. For example amplification of myc can cause cancer this is often observed with fluorescence in situ hybridisation

13

Give an example of where translocation the same gene in a different place can be oncogenic

In Burkitt’s lymphoma there is a chromosomal translocation between IgH and myc. The results in the myc gene relocated downstream of IgH promoter which drives high expression of the transcription factor. This results in amplification of the gene which in turn causes the cancer

14

Describe how the southern blot that identified cRas was able to utilise a HRas probe

You can define how stringent the probe in southern blotting will be for the DNA. It turns out that HRas and cRas are homologous and this is why the HRas probe can bind to cRas DNA in the southern blot

15

Other than glycine 12 what other residues in proto-oncogenic cRas can be mutated to cause the transition to an oncogene

Amino acid substitution of glutamine 61

16

What was the significance of identifying the proto-oncogene cRas to be almost identical to the viral oncogene HRas in terms of how viral oncogenes evolved

The high degree of homology between cRas and HRas is most likely because the virus had picked up the cellular Ras gene from a previous host cell. This had been extracted from the DNA along with the virus due to the ability of the virus to spontaneously leave host genome bringing host genetic information with it

17

What is the clinical significance of oncogenes

Oncogenes can be used to give an indication of disease prognosis. For example patients with HER2 amplifications of greater than 5 in breast cancer biopsies will often show less than a 50% survival rate over 5years. In comparison breast cancer patients without HER2 amplifications often have a 2-3 better chance of survival

18

What is the additional gene found in RSV that confers its ability as an oncogenic virus

The sarcoma gene (src)

19

What was the contribution of Temin and Rubin to the knowledge of Rous Sarcoma Virus (RSV)

Temin and Rubin infected cells with RSV and noticed that this resulted in a productive life cycle. In addition these cells showed traits similar to cancerous cells. This was the first in vitro model for RSV induced sarcomas

20

What evidence lead to the idea that that chemicals and radiation induced provirus expression

Treating non-viral infected cells with the nucleotide precursor 5-bromo-2-deoxyuridine leads to the induction of provirus expression

21

A Proof that the mutation alone can cause transformation via the proto-oncogene to oncogene transition T or F

T

22

Describe how the proto-oncogene cellular Ras (cRas) was originally identified

NIH3T3 cells were transformed with a mutagen. The genomic DNA from these transformed cells was then electrophoresed and transferred to a nitrocellulose membrane. The DNA was then interrogated with a radioactively labelled probe (Southern blotting). The probe used was a H-Ras viral oncogene probe. HRas was an already characterised viral oncogene so was used as a probe to see if there was a homologous gene found in the cells. Indeed there was a positive staining for an oncogene in the cells that had been transformed with the mutagen than those that had not. This was cellular Ras or c-Ras the first cellular proto-oncogene

23

Only point mutations can convert proto-oncogenes to oncogenes T or F

F – oncogene can also arise from structural deletions changes in regulation amplification and translocations

24

What wrong assumption was made on the basis that some chemicals could induce the expression of proviral elements

That a central idea to tumorigenesis was the activation of a latent provirus that was present in all of the host cells

25

Discuss the significance of anchorage independent growth in cancerous cells

Healthy cells in a semi-solid medium such as agarose or methylcellulose form cell sheets. However cancerous cells that have been infected with virus’s form foci or clusters of cells. This is an indication of anchorage independent growth were the cells don’t need to adhere to the medium to grow. This is now used as an assay for viral tumour induction

26

Bob Weinberg’s experiments provided the idea of oncogenes that cause transformation and cancer in tumour cells. What reverse conclusion was also indicated from his work

That normal cells must contain genes capable of becoming oncogenes these were referred to as proto-oncogenes

27

What was the contribution of Peyton Rous to the viral model of cancer

Peyton Rous discovered the Rous sarcoma virus. He removed sarcomas from adult chickens and ground up the tumours in a fine sand column filter. This ensured that the filtrate was sterile before he injected it into a young tumour free chicken to see then effects. This ultimately resulted in sarcoma development in these chickens. This was the first evidence for a transmissible element involved in the formation of a sarcoma

28

Describe the experiments carried out by Bob Weinberg to determine the number of genetic changes required to cause transformation

Weinberg treated virus-free-cells (C3H10T1/2 cell line) with 3-methylcholanthrene a mutagen which causes transformation. The DNA from these cells was then extracted a process which fragmented the chromosomes. These chromosome fragments were then CaPO4 transfected into normal fibroblasts. These transfected fibroblasts subsequently formed foci in agar indicative of transformation. The foci from these cultures were then injected into nude mice subsequently generating tumours. The mutagenicity of methylcholanthrene is such that it is unlikely that more than one gene was mutated using this mutagen. Calculations revealed that using the transfection method would result in each cell taking up ~0.1% of the total genome from the chromosome fragments that were transfected into them. This corresponds to around 30 genes and combined with the idea that the rate of methylcholanthrene induced mutagenesis meant that it was unlikely that more than one of these genes were mutated there must be specific singe genes that when mutated cause cancer. This lead to the idea of oncogenes

29

Which Ras isoforms are specifically viral oncogenes

HRas and KRas (Harvey and Kirsten murine sarcoma viruses)

30

Outline the genome of the RSV virus

RSV is a retrovirus with a diploid RNA genome. It consists of only 4 genes; and envelope (env) core protein (gag) reverse transcriptase (pol) and src