FA23 week 3 Problem Set w-correction to 1b and 7a (2)

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1 BICD100 FA23 Week 3 Problem Set Question numbers are color coded to show which ones are based on Monday lecture material vs. Wednesday or Monday + Wednesday lecture material 1). The RNA sequence of the first six codons of a gene are: AUG AGA GAA UGG ACG AUG Use the codon table on textbook pg. 533 to answer the following questions. a. What amino acid sequence is encoded by these six codons? Met-Arg-Glu-Trp-Thr-Met b. Which of the following could introduce a stop codon that truncates this polypeptide? For each one you select (i.e. each one where your answer is yes), write the altered 18 nucleotide RNA sequence, show what was changed/added/deleted, and underline the new stop codon. i. Point mutation/substitution altering codon 3: AUG AGA UAA UGG ACG AUG ii. Point mutation/substitution altering codon 5: AUG AGA GAA UGG AUG AUG iii. Insertion of 3 nucleotides between codons 3 and 4: AUG AGA GAA UAG UGG ACG AUG iv. Insertion of 1 nucleotide between codons 1 and 2: AUG UAG A GAA UGG ACG AUG v. Deletion of the first 2 nucleotides of codon 3: AUG AGA A UG G ACG AUG c. Which of the mutations listed in part b, above, creates a frameshift? iii iv and v d. Which of the mutations listed in part b, above, are likely to be obtained via CRISPR-Cas9 genome editing of this sequence? i and ii What DNA repair mechanism would be involved in creating the mutation type(s) you identified in part d, above? non-homologous end joining (NHEJ). NHEJ is a repair mechanism that joins together broken ends of DNA without using a template. This can sometimes lead to the insertion or deletion of nucleotides, which can result in point mutations. In addition to the mutations listed above, there are other types of mutations that could introduce a stop codon into this polypeptide. For example, a large deletion that removes a portion of the coding sequence could result in a frameshift mutation that creates a premature stop codon. e. 2). IL2RG encodes a subunit of a cytokine receptor that plays a role in the development of the immune system. Mutations in this gene cause approximately 30% of inherited immunodeficiency conditions (severe combined immunodeficiency disorder or SCID) in humans. The IL2RG gene is composed of 8 exons. Assume that all the mutations described below cause loss of gene/protein function. For each of the following pairs of mutations, indicate which one you think is more likely to be a null and which a leaky allele, or even a silent mutation if applicable (causing no disease). You will want to consult a codon

2 table for some of these. Explain your reasoning for each answer in 1-2 sentences, including use of appropriate terminology such as silent, nonsense, missense, frameshift, SNP, indel, etc. a. Single nucleotide insertion in exon 1 vs. exon 7: Most likely a null allele because exon 7 contains the stop codon, so an insertion is likely to cause a frameshift mutation, which will result in a truncated and non functional protein. Insertion in exon 1 may or may not cause a frameshift mutation, depending on the location of the insertion relative to the reading frame. However, it is more likely to disrupt the reading frame and cause a null allele than an insertion in exon 7. cont’d b. Deletion of two vs. three nucleotides in exon 4: More likely to be a leaky allele because a deletion of two nucleotides is less likely to cause a frameshift mutation than a deletion of three nucleotides. If the deletion does not cause a frameshift mutation, it may only result in replacement of a single amino acid in the protein. c. Single nucleotide change in a codon in exon 1 from UAU to UAC vs. UAG: silent mutation because UAC and UAU both encode amino acid Tyrosine. d. An inversion of exons 2-5 vs. an inversion of part of the cis-regulatory module that reduces but does not eliminate expression of this gene: Null allele because an inversion of exons 2-5 will disrupt the reading frame and cause a frameshift mutation, which will result in a truncated and non-functional protein. 3). Which of the mutations described in question 2, if any, is/are likely to be the result of each of the following types of DNA damage or mutagen exposure? Refer to letters a-d used in Q2. a. a base mispairing event during DNA replication: single nucleotide change from a base mispairing event during DNA replication such as in 2c. b. UV irradiation: Pyrimidine dimer caused by UV radiation, blocking DNA replication and transcription and leads to mutations. c. X-ray irradiation: Double stranded break- can be repaired, but errors in repair can lead to mutations such as del, inserts, and inversions. 4). Which of the mutations described in question 2 (if any) could have been repaired via… a. mismatch repair: repair a single nucleotide insertion

3 b. proofreading, i.e. the 3’ exonuclease activity of DNA polymerase : repair a single nucleotide insertion c. nucleotide excision repair: repair a pyramidine dimer 5). A geneticist uses CRISPR-Cas9 editing to introduce mutations into a gene containing the following sequence in one of its exons. The CCG in red is a “PAM” sequence that is required for Cas9 cleavage, and the underlined portion is targeted for cleavage by the guide RNA: One of the mutant alleles obtained has a 4bp deletion as illustrated here: b. Draw the double-strand DNA molecule, containing the complete 28 nucleotide sequence shown at the top, after it was cut by Cas9 showing the overhangs at both ends of the cut that were trimmed off prior to NHEJ to create this 4bp deletion. Highlight the overhanging sequences that get trimmed off. 5'-GCGCCGTCCCCTGTCAGGACACTTCA-3' | (Cas9 cut) | 5'-GCGCCG CTGTCAGGCGACACTTCA-3' ^^^^^^ ^^^^^^ (4 bp deletion)

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4 6). Consider the three mutations described below (i-iii) and then answer questions a-d. i. One of the mutations causing cystic fibrosis in humans is a deletion of three nucleotides that eliminates a phenylalanine at position 508 of the CFTR protein ( ∆ 508). Normally, CFTR protein is localized to the plasma membrane, where it functions as a chloride ion channel. ∆ 508 CFTR is misfolded and all of it is degraded without ever reaching the cell surface. ii. The yeast transcription factor Gal4p contains a DNA-binding domain and a transcriptional activation domain. An allele with a deletion the gene portion encoding the activation domain encodes a truncated Gal4p containing only the DNA-binding domain. It binds to Gal4p target genes at appropriate binding sites in their upstream regulatory regions, but does not activate their transcription. In cells with both wild type and mutant forms of Gal4p, the truncated Gal4p binds more efficiently to target DNA sequences than wild type. iii. Rhodopsin is a protein made in photoreceptor cells of the retina, which detect light to generate visual cues. A missense mutation in the rhodopsin gene produces a rhodopsin protein that is activate all the time, whether or not the eye is exposed to light, resulting in constant light perception. a. Which of these mutations is most likely to be a null allele and why? Cystic fibrosis mutation (A508 deletion) is most likely to be a null loss of function mutation. b. Which of these mutations is mostly likely to be a gain of function mutation and why? Rhodopsin gene missense mutation (constitutively active) is most likely to be a gain of function mutation. c. Which of these mutations is mostly likely to be a dominant negative mutation and why? Gal4p transcription factor mutation (activation domain deletion) is most likely to be a dominant negative mutation due to its interference with wild-type protein function. d. For which of these mutations would observation of an abnormal/disease phenotype in heterozygotes indicate that the gene is haploinsufficient? Observation of an abnormal/disease phenotype in heterozygous individuals of the Gal4p transcription factor mutation (activation domain deletion) and Acid maltase gene mutation (intron-exon boundary change) could indicate haploinsufficiency. 7). APC (adenomatous polyposis coli) is a tumor suppressor gene that is almost always mutated in colon cancer (colorectal carcinoma). Most people inherit two wild type copies of APC, but in rare cases a mutant copy is inherited, causing the formation of abundant colon polyps and a very high lifetime risk of colon cancer – a condition called familial adenomatous polyposis.

5 a. Consider the results described below of APC allele analysis in two biopsy samples from a person with FAP. Which of these results do you think is for skin cells, and which for colon cancer cells? Provide 1-2 sentences of rationale for each answer that includes a statement about the type(s) of mutation(s) observed and whether they are most likely gain or loss of function alleles. i. One allele has a 1bp insertion in the coding region near the beginning of the gene; the other is wild type. This 1bp insertion near the beginning of the gene suggests a loss-of-function mutation. It disrupts the normal reading frame and function of the gene, reducing its tumor-suppressing abilities. Cont.’d ii. One allele has a 1bp insertion in the coding region near the beginning of the gene; the other has a deletion removing half the coding sequence. The 1bp insertion disrupts the function of one allele, and the deletion of half the coding sequence is a severe loss-of-function mutation. The loss-of-function allele is complemented by the gain of function in the other allele, which contributes to uncontrolled cell growth and tumor formation. b. Are either of the following results expected from an analysis of APC alleles in tumor cells of a person with “sporadic” colon cancer, i.e. someone who did not inherit an APC mutant allele (does not have FAP)? For each answer, provide a single sentence of rationale for why it is or isn’t in line with expectations. i. One allele has a mutation that massively increases the expression level of the gene and the other one is wild type. ii. Both alleles have a deletion removing half the coding sequence (tumor cells are homozygous for this mutation). The remaining questions (on the next page) are based on the background information below that refers to this drawing:

6 Drosophila Patched ( PTC ), Smoothened ( SMO ) and Hedgehog (HH), and their orthologs in many other species, play important roles in development. HH encodes a secreted protein that binds to a transmembrane receptor on the cell surface encoded by PTC . PTC protein interacts with another transmembrane protein with signaling properties encoded by the SMO gene. In the absence of HH (left, above), interaction with PTC inhibits the signaling activity of SMO protein. Binding of HH to PTC (right, above) relieves PTC inhibition of SMO, leading to SMO signaling and activation of downstream targets that can include genes promoting cell division, depending on the cellular context. Basal cell carcinomas (BCCs) are skin cancers representing at least one third of all cancers diagnosed in the U.S. each year. The frequency of BCCs is increased by exposure to sunlight.

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7 8). Loss of function mutations in a human homolog of PTC , PTCH2 , are frequently found in BCCs. a. Together with the background information above, does this indicate that the wild type version of human PTCH2 is a tumor suppressor gene, proto-oncogene, or oncogene? b. People with a commonly occurring, wild type allele of PTCH2 with two adjacent thymines at a particular site in the coding sequence are more prone to BCCs than people without this allele. What explanation would you propose for this (in one sentence)? c. The “two adjacent thymines” allele of PTCH2 causes a bigger increase in BCC risk for people with xeroderma pigmentosum (XP), who lack components of the nucleotide excision repair pathway, compared to people without XP. What explanation would you propose for this (in one sentence)? d. About 0.5% of people who get a BCC have a heritable condition called Gorlin Syndrome, characterized by very frequent BCCs and other phenotypes. This condition is due to inheritance of a single, non-functional allele of PTCH2 . How would you explain why people with Gorlin Syndrome get BCCs so much more often than people without this condition (in one sentence)? 9). Mutations in SMO are also common driver mutations in BCC. The SMO mutation found most commonly in BCCs changes a Trytophan at position 535 in the SMO protein to a Leucine (W535L). The W535L version of SMO protein has HH-independent signaling activity - it activates downstream target genes constitutively. a. Use a codon table to deduce the DNA sequence change producing the W535L allele of SMO , assuming that only one nucleotide changes: wild type codon:_______ mutant codon: ________ b. Is the W535L mutation a gain or loss of function mutation? c. Is wild-type SMO a proto-oncogene, a tumor suppressor gene or an oncogene?

FA23 week 3 Problem Set w-correction to 1b and 7a (2)

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