Quiz 11

*p53 is a transcription factor whose function is lost in many cancers. MDM2 binds to the transcriptional activation domain of p53, thereby inhibiting the p53 function; MDM2 also has an ubiquitinating activity that causes proteasome-mediated degradation of p53. Is MDM2 a tumor suppressor or a proto-oncogene?* Proto-oncogene Tumor Suppressor

Proto-oncogene

*The MAP kinase pathway is activated by ligand binding to a transmembrane receptor, which switches Ras on via GEF (Guanine nucleotide Exchange Factor) that counteracts GAP (GTPase Activating Protein) activity. Ras-GTP in turn activates the downstream signaling intermediates (kinases), which are activated by phosphorylation. Which drug CANNOT be used to target cancer cells that have elevated receptor activation?* Small molecule inhibitors that interfere with GEF activity Small molecule inhibitors that bind the ATP-binding pocket of kinases Antibodies that block the ligand-binding domain of receptor Antibodies specific for ligand to prevent receptor binding Small molecule inhibitors that interfere with GAP activity

Small molecule inhibitors that interfere with GAP activity

*Five to ten percent of all breast cancers, which can affect both females and males (to a lesser extent), have inactivating mutations in genes BRCA1 or BRCA2, which are important players in DNA double-strand break repair. Spectral karyotyping (a chromosome "painting" technique) of BRCA1- and BRCA2-mutant cells reveals gross chromosomal rearrangements such as translocations and deletions, as well as fusions. Is the BRCA gene a tumor suppressor or a proto-oncogene?* *And which of these results would support a sporadic origin of breast cancer? (Assume there is no additional cancer.)* *Select two that apply.* Both blood cells and tumor cells are homozygous wild type. Both blood cells and tumor cells are homozygous mutant. Proto-oncogene Blood cells are heterozygous and tumor cells are homozygous mutant. Tumor suppressor Blood cells are homozygous wild type and tumor cells are homozygous mutant.

Tumor suppressor Blood cells are homozygous wild type and tumor cells are homozygous mutant.

*How can you simultaneously target multiple genes for knockout using CRISPR?* Use multiple guide RNAs (sgRNAs). Inject into multiple cell types. Use multiple donor DNAs. Use multiple Cas9, each cloned into a specific regulatory region.

Use multiple guide RNAs (sgRNAs).

*Huntington disease is a dominant disease caused by expansion of the trinucleotide repeat region of the Htt gene that results in the production of a Huntingtin protein with an expanded number of glutamines. An animal model with most features of this syndrome could be created by* knocking in a wild-type copy of the Htt gene to a mouse or primate genome. randomly inserting a transgene containing a wild-type allele of the Htt gene to a mouse or primate genome. randomly inserting a transgene containing a disease-causing mutant allele of the Htt gene to a mouse or primate genome knocking out one copy of the wild-type Htt gene from a mouse or primate genome. knocking out both copies of the wild-type Htt gene from a mouse or primate genome.

randomly inserting a transgene containing a disease-causing mutant allele of the Htt gene to a mouse or primate genome

*How can a mutation in a tumor suppressor behave as a recessive allele at the cellular level but appear as a dominant allele in pedigree analysis?* At the cellular level both alleles need to be mutated for an en effect, while in a pedigree the cancer may be dominantly inherited because of the high frequency of the mutant allele in a population, increasing the chance of producing a homozygous mutant child. At the cellular level both alleles need to be mutated for an en effect, while in a pedigree the cancer may be dominantly inherited because the majority of tumor suppressors are haploinsufficient. At the cellular level both alleles need to be mutated for an en effect, while in a pedigree the cancer may be dominantly inherited due to frequent loss/inactivation of the normal allele. At the cellular level both alleles need to be mutated for an en effect, while in a pedigree the cancer may be dominantly inherited due to gain of function mutation in a tumor suppressor.

At the cellular level both alleles need to be mutated for an en effect, while in a pedigree the cancer may be dominantly inherited due to frequent loss/inactivation of the normal allele.

*p53 responds to diverse stress signals by orchestrating specific cellular responses, including transient cell cycle arrest (through activation of p21, a CDK inhibitor), DNA repair, and apoptosis. Which of the following is most likely to occur from inactivation of p53? (Select two that apply.)* Cells undergo apoptosis due to accumulation of DNA damage Cells have a high number of point mutations and fragments of chromosomal DNA lacking telomeres and a centromere Cells are defective in the G1-to-S checkpoint due to increased phosphorylation of Rb Cells arrest in G1 phase due to decreased phosphorylation of Rb

Cells have a high number of point mutations and fragments of chromosomal DNA lacking telomeres and a centromere Cells are defective in the G1-to-S checkpoint due to increased phosphorylation of Rb

*What is a feature unique to adeno-associated viral (AAV) vectors and is not shared with retroviral vectors?* Packaging the therapeutic DNA into virus-like particles requires "helper DNA" which expresses all the proteins required to make viral particles. Therapeutic gene is transcribed and translated in patient cells using the host transcription and translation machinery. Packaging cells do not make active viruses because helper DNA lacks packaging sequences and therapeutic DNA lacks essential viral genes. Therapeutic gene integrates randomly into the patient's genome. Gene therapy needs to be repeated periodically to provide a constant supply of the therapy protein.

Gene therapy needs to be repeated periodically to provide a constant supply of the therapy protein.

*Angelina Jolie's mother and maternal grandmother died of ovarian cancer. Her maternal aunt died of breast cancer. Angelina Jolie opted to have a blood test to see if she had a BRCA1 mutation and found that one copy of the gene was wild type, and the other copy had a nonsense mutation that would cause production of a nonfunctional protein.* *Is the tumor-causing mutation dominant or recessive to the wild type at the cellular level? And what is the genotype of the non-tumor cells of her mother, maternal aunt, and maternal grandmother? (Choose two that apply.)* Mutation is recessive to wild-type. Heterozygous Homozygous wild type Homozygous mutant Mutation is dominant to wild-type.

Mutation is recessive to wild-type. Heterozygous

*The following diagram shows the complex between an sgRNA and a target site in the genome where Cas9 cuts DNA at the indicated positions. The protospacer adjacent motif or PAM site (5'-NGG-3' on one strand; N is any base) needs to be present adjacent to the target site in order for Cas9-mediated cleavage to occur. One problem associated with CRISPR/Cas9 technology is off-target effects. Part of the reason is that single base-pair mismatches between the target site and the sgRNA in the 20 bp DNA/RNA hybrid do not prevent Cas9 cleavage of the target site. What strategy do you need to use in order to target the mutant Htt allele that causes Huntington disease without off-target effects?* Look for a unique target sequence in the Htt gene that lacks a PAM site nearby. Use mutant Cas 9 enzyme with decreased target specificity. Make sure that no sequences in the genome are identical or too similar to the 20 bp sequence involved in base pairing between the sgRNA and the target site. Use multiple sgRNAs, each of which has one mismatch with the target sequence at different positions. Design sgRNA that can base pair with several regions in the genome including the Htt gene locus to ensure efficient cleavage of the mutant allele.

Make sure that no sequences in the genome are identical or too similar to the 20 bp sequence involved in base pairing between the sgRNA and the target site.