Lecture 2 Cellular Oncogenes Flashcards
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
The G12V transition in cRas is sufficient to create an oncogene T or F
T – this is sufficient to cause tumours in nude mice
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
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).
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.
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.
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
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
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
How are myc genes referred to when they are present in a viral genome
V-myc
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.
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
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
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
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
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
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
What is the additional gene found in RSV that confers its ability as an oncogenic virus
The sarcoma gene (src)
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
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
A Proof that the mutation alone can cause transformation via the proto-oncogene to oncogene transition T or F
T
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
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
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
