3). Consider the four mutations (i-iv) described below: 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 (D508). Normally, CFTR protein is localized to the plasma membrane, where it functions as a chloride ion channel. D508 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. Mutations in the acid maltase gene in humans cause a metabolic disorder where glycogen over- accumulates in multiple tissues. In individuals with a relatively mild form of the disease, a single base pair change at an intron-exon boundary interferes with splicing most, but not all of the time; nozygous mutant cells have about 10% of normal enzyme activity. iv. 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 mostly likely to be a dominant negative mutation and why? b. Which of these is most likely to be a null loss of function mutation and why? c. Which of these is most likely to be a leaky loss of function mutation and why? d. Which of these is mostly likely to be a gain of function mutation and why?

1. With reference to the figure, discuss the results of the Western Blotting experiments, what they tell you about the behaviour of the estrogen receptor, and how this relates to its known biological activity.  2. Propose what suitable controls should be included for this experiment and explain what you would expect to see as results from these controls in the Western Blot in the figure.

1. "Interferons (IFNs)‐α and ‐β are expressed in response to a virus infection and are released from the cell in which they are produced. IFNs induce an antiviral state in other neighboring cells. a. Which cellular process is inactivated when IFN‐treated cells are infected with a virus? b. One arm of the IFN‐induced antiviral state is the synthesis of 2′,5′‐oligoA in response to viral infection. In one sentence or a simple diagram, what is the effect of this on the cell? c. Another arm of the IFN‐induced antiviral state is activation of the protein kinase in response to viral infection. In one sentence or a simple diagram, what is the effect of this on the cell? d. All cells contain the genes for IFNs. IFN synthesis is stimulated by virus infection. Would you expect a cell that has been treated with IFN to synthesize IFN in response to a viral infection? Explain your answer."

Wilms tumor 1, or nephroblastoma, is caused by mutations in the WT1 gene, which encodes a transcription factor. You have identified a novel variant in WT1: Arg422Pro.   You have control cells and cells that have been engineered to carry the homozygous WT1 p.Arg422Pro mutation. You want to assess effects of this mutation on a variety of endpoints. For each endpoint listed below, choose the one technique is best suited to answer the question.   Choose from: array CGH, qRT-PCR, qPCR, RNA-seq, FISH, in situ hybridization, western blot, immunostaining, WT1 ChIP-seq, WT1 ChIP-PCR, ATAC-seq, 3C     Endpoint Technique? WT1 protein amount (quantitative) Western blot WT1 protein binding to all enhancers, genome-wide Chip-seq WT1 mRNA amount (quantitative)   WT1 protein subcellular localization   Quantitative assessment of all mRNAs in these cells (genome-wide) RNAseq Chromatin interactions between a specific WT1 chromatin binding site (identified above)…

Many neurodegenrative diseases are thought to be caused by increased levels of unfolded proteins. Which of the following conditions would you expect to increase unfolded protein levels (select all that apply)?   A. Reduced expression of chaperone proteins   B. A mutation that inactivates the proteasome   C. Increased levels of ubiquitin   D. Inactivating mutation in ubiquitin ligase, an enzyme necessary for attaching ubiquitin to proteins in cytosol   E. Mutation of N-terminal amino acid from Methionine to Arginine

Drug X shown below is a kinase inhibitor used to treat multiple types of breast cancer. Breast cancer cells are treated with the drug and after 8 hr are lysed and the presence of various proteins is shown by a band on a western blot. The presence of the phosphorylated form of the protein is shown by the presence of a band when probed with antibodies that recognize the phosphorylated form of protein and are indicated by a p- in front of protein name (e.g. p-AKT). Based on this information, which kinase is likely the target and is inhibited by Justify your answer in 3-4 sentences. Drug X. F FF N. -N S IZ H₂N `N No Drug (+) Drug p-AKT AKT p-GSK3B GSK3B p-p70S6K p70S6K

Ferritin levels are controlled in cells to ensure that free iron levels do not surpass a toxic threshold. Its levels must never be too high or it will titrate too much iron and jeapardize critical cellular iron-dependent enzyme activities. How is this achieved? Choose one. a) Elements present in the 5' UTR contribute to its translational block in low iron conditions. b) The Ferritin mRNA is under IRE-BP control so that in high iron conditions IRE-BP is not in its active conformation an has no negative effect on Ferritin mRNA translation c) IRE-BP can effectively bind to destablizing elements in the 3'UTR of the Ferritin mRNA, thus drastically reducing the half life of the transcript 4) a and b only 5) a, b and c

Describe how transcription would be affected in the Galactose metabolizing pathway in Yeast in the presence of the following mutations. 1. A mutation that resulted in an inability of Gal80 to enter the nucleus. 2. A mutation that resulted in a lack of ability of Gal3 to bind galactose.

4). p53 (sometimes called TP53 for “tumor protein 53") is a human tumor suppressor gene that is mutated in the majority of human cancers (many tumor types). a. For each of the mutations described below (i-iv): is this a mutation you would expect to find when sequencing p53 alleles from tumor cells? Why or why not? i. A missense mutation encoding a hyperactive form of the protein. ii. A deletion of the gene. iii. An insertion in the promoter that increases transcription 10-fold. iv. A nonsense mutation. b. When sequencing the p53 gene in tumor cells, would you expect to find only mutant version(s) of the gene or a mix of mutant and wild type versions? c. For any of the mutations you said you would expect to find in tumor cells, would you expect tumor cells to be homozygous (same mutation on both chromosomes)? Why or why not? d. Individuals with Li-Fraumeni syndrome have a very high risk of tumors originating in various tissues due to inheritance of a loss-of-function mutant allele of…

2. Below, recognition sites of two of these enzymes, Pstl and Nsil, are given 5'... CTGC AG. 3... GACGTC...5 3 Pstl recognition and cleavage site 5... ATGCAT 3'... TACGTA ... 5' 3' ... Nsil recognition and cleavage site a. Does cleavage by Pstl result in a 5' or 3' overhang? What is the sequence of this overhang? b. Does cleavage by Nsil result in a 5' or 3' overhang? What is the sequence of this overhang? c. Suppose you have a cloning vector that contains a Pstl recognition site and you also have foreign DNA that was cut with Nsil. Can this DNA be ligated into the Pstl site of the vector, and if so, explain its reason and draw the new ligation site. d. Can the new ligated DNA segment sequence be cut from the vector with Pstl or Nsil ? What potential problems do you see?